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Regulation of Intracellular Calcium Concentrations by Calcium and Magnesium in Cardioplegic Solutions Protects Rat Neonatal Myocytes from Simulated Ischemia
J Mol Cell Cardiol 30, 1105–1114 (1998) Article No. mc980676
Regulation of Intracellular Calcium Concentrations by Calcium and Magnesium in Cardioplegic Solutions Protects Rat Neonatal Myocytes from Simulated Ischemia Takashi Ichiba, Naruto Matsuda, Naoaki Takemoto, Shingo Ishiguro, Hiroaki Kuroda and Tohru Mori Department of Surgery, Tottori University, Faculty of Medicine, Yonago, Japan (Received 19 November 1997, accepted in revised form 23 February 1998) T. I, N. M, N. T, S. I, H. K T. M. Regulation of Intracellular Calcium Concentrations by Calcium and Magnesium in Cardioplegic Solutions Protects Rat Neonatal Myocytes from Simulated Ischemia. Journal of Molecular and Cellular Cardiology (1998) 30, 1105–1114. The effects of calcium and magnesium ions in cardioplegic solutions on cardioprotection and intracellular calcium ion handling during ischemia and reoxygenation were investigated in cultured neonatal rat myocardial cells. Myocytes were subjected to simulated ischemia for 60 min at 37°C in hyperkalemic cardioplegic solutions containing various concentrations of calcium and magnesium ions, followed by 30 min of reoxygenation. For each Ca2+ concentration (0.1, 0.6, 1.2, or 2.4 m), the Mg2+ concentration was either 0, 1.2, 8, or 16 m. The increase in intracellular Ca2+ concentration during ischemia and reoxygenation was suppressed by the addition of magnesium ion, independent of cardioplegic Ca2+ concentration. The recovery of spontaneous contraction rate and enzyme leakage (creatine phosphokinase and lactate dehydrogenase) during both ischemia and reoxygenation correlated with the degree of inhibition of intracellular Ca2+ accumulation. However, in the 0.1 m Ca2+ groups in which the Mg2+ concentration was greater than 8 m, the intracellular Ca2+ concentration increased during reoxygenation in a dose-dependent fashion of Mg2+, and was associated with increased enzyme leakage. The findings suggest that in immature cardiac myocytes, the concentrations of Ca2+ and Mg2+ present in cardioplegic solutions control the intracellular Ca2+ concentration during ischemia and reoxygenation, which, in turn, influences the cardioprotective effect of the cardioplegic solution. 1998 Academic Press K W: Cardioplegic solution; Simulated ischemia; Intracellular calcium; Fluo-3; Cell injury; Immature myocytes; Calcium ion; Magnesium ion.
Introduction Abnormal handling of intracellular calcium ions (Ca2+) during ischemia and reperfusion is one of the principal causes of post-ischemic myocardial dysfunction. Increased intracellular Ca2+ stimulates a number of calcium-dependent enzymes, including phospholipases and proteases, which are potentially
harmful to the cell (Nayler and Elz, 1986; Otani et al., 1989). The use of hyperkalemic cardioplegic solutions (CP) attenuates the increase in the intracellular Ca2+ concentration ([Ca2+]i) that occurs during ischemia and improves post-ischemic contractile function (Ataka et al., 1993). The individual components of CP have been studied in an attempt to optimize the inhibition of intracellular Ca2+
Please address all correspondence to: Takashi Ichiba, Department of Surgery, Tottori University Faculty of Medicine, 36-1 Nishi-machi, Yonago, Tottori 683, Japan.
0022–2828/98/061105+10 $30.00/0
1998 Academic Press
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accumulation and thereby improve myocardial protection. The concentrations of Ca2+ and magnesium ions (Mg2+) in cardioplegic solutions have been found to be important in myocardial protection (Brown et al., 1991; Ataka et al., 1993; Takemoto et al., 1994). Specifically, the extracellular Ca2+ concentration ([Ca2+]o) alters the amount of Ca2+ bound to the sarcolemma, thus determining the magnitude of Ca2+ influx (Langer, 1984). Further, Mg2+ acts as a physiologic Ca2+ channel blocker (Iseri and French, 1984), decreasing Ca2+ influx into the cell. Previous studies of Ca2+ and Mg2+ metabolism have not adequately examined the responses of the immature heart. In recent studies, it has been shown that Ca2+ handling in immature myocardial cells differs greatly from that in the mature myocyte. The difference arises from the fact that an immature myocyte possesses little sarcoplasmic reticulum (SR), and regulates intracellular Ca2+ predominantly at the level of the sarcolemma. In contrast, a mature myocyte relies largely on its developed SR to control intracellular Ca2+ (Hoerter et al., 1981; Maylie, 1982; Nakanishi and Jarmakani, 1984). Therefore, the effects of extracellular Ca2+ and Mg2+, which control Ca2+ influx via the sarcolemma, may be more important for immature hearts than mature hearts. In the present report, neonatal, cultured myocardial cells were used to determine the effects of Ca2+ and Mg2+ in hyperkalemic CP on the regulation of intracellular Ca2+ and myocardial protection during both ischemia and reoxygenation.
Cell culture Cultured neonatal rat myocardial cells were prepared by a modification of the method used by Simpson and Savion (1982). In brief, ventricles from 2- to 3-day-old Wistar rats were minced and dissociated at 37°C using 0.1% collagenase. The digestion medium was discarded, and the cells were resuspended in culture medium (M199), supplemented with 4% FBS, 100 U/ml penicillin, 100 lg/ml streptomycin, 10 lg/ml transferrin, and 10 lg/ml insulin. The cell suspension was preplated on a 60-mm diameter plastic dish for 30 min at 37°C in a CO2 incubator. The non-attached, viable cells were seeded onto type I collagen-coated 35mm dishes (2×105 cells per dish) and incubated for 3 days prior to use.
Cell loading with Fluo-3 Fluo-3 AM was used to determine [Ca2+]i. Myocardial cells were incubated in M199 containing 10 l Fluo-3 AM for 30 min at 37°C. Extracellular Fluo-3 AM was then removed by changing the medium twice with M199. This loading period was followed by a 20-min post-incubation period at 37°C, which allowed complete hydrolysis of the Fluo-3 AM ester.
[Ca2+]i measurements
Materials and Methods Materials Reagents used in this study included: collagenase, transferrin (Wako Chemical, Osaka, Japan), medium 199 (M199), penicillin, streptomycin (BioWhittaker, Walkersville, MD, USA), insulin (Becton Dickinson, Two Oak Park, MA, USA), fetal bovine serum (FBS) (Cansera, Ontario, Canada), the acetoxymethyl ester Fluo-3 (Fluo-3 AM) (Dojindo, Kumamoto, Japan), amytal, carbonyl cyanide mchlorophenylhydrazone (CCCP) (Sigma, St Louis, MO, USA), ionomycin (Calbiochem-Novabiochem, La Jolla, CA, USA), and calcium calibration buffer kit II (Molecular Probes, Eugene, OR, USA). Rats were treated in accordance with the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals.
We employed a confocal laser scanning system (INSIGHT PLUS, Meridian Instruments, Okemos, MI, USA) equipped with an Olympus IMT-2 inverted microscope to measure [Ca2+]i. Intracellular Fluo3 was excited at 488 nm, and its emission fluorescence was measured at 530 nm. The fluorescence images were recorded using a CCD camera equipped with an intensifier and stored on video tape (30 frames per s). In order to minimize photobleaching, the fluorescence acquisition was performed for 3 s at 5-min intervals. In between data acquisition, the cells were not exposed to the exciting wavelength light. The data were analysed using INSIGHT-IQ software. The diastolic fluorescence values during a 3-s period were averaged for individual cells. In preliminary experiments, the fluorescence intensity of Fluo-3 did not decrease significantly after 120 min (data not shown).
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Beating rate recovery measurements The beating rate of synchronized myocytes was determined by viewing the cultured cells microscopically. The beating rate per min was recorded prior to ischemic period and after 30 min of reoxygenation that followed the ischemic period. The recovery ratio for the beating rate during reoxygenation is expressed as a percentage of the beating rate prior to ischemic period.
Enzyme leakage determination The media used during ischemic and reoxygenated period were assayed spectrophotometrically to determine the quantity of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) released from cells. The enzyme activities were determined using the uv-rate and Wro´blewski–La Due methods employing a uv-visible recording spectrophotometer (UV-160, Simadzu, Kyoto, Japan). The total amounts of CPK and LDH released were expressed as international milli units (mIU) per dish.
Experimental protocol
Pre-ischemic period Cultured myocardial cells were placed in a temperature-controlled (37°C) chamber mounted on the stage of an inverted microscope. A pair of platinum stimulating electrodes were placed 2-cm apart in the chamber. The myocytes were first incubated for 30 min with Tyrode solution containing: 140 m NaCl, 6 m KCl, 1 m CaCl2, 10 m glucose, and 5 m N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) (Macleod, 1989). The medium was equilibrated with 100% O2 at 37°C, and the pH was adjusted to 7.4 with NaOH. The beating rate and fluorescence of Fluo-3 bound to intracellular Ca2+ were recorded as control values during the last 1 min of this period.
Ischemic period Myocytes were then subjected to chemically-induced hypoxic, glucose-free perfusion simulating ischemia for 60 min at 37°C. The solutions (cardioplegic solutions; CP) contained: 110 m NaCl, 16 m KCl, 5 m HEPES, 2 m sodium cyanide to inhibit oxidative phosphorylation, and varying concentrations of CaCl2 and MgCl2. The pH of these
solutions were adjusted to 7.4 with NaOH. The [Ca2+]o was either 0.1, 0.6, 1.2, or 2.4 m. For each [Ca2+]o, the Mg2+ concentration ([Mg2+]o) was 0, 1.2, 8, or 16 m.
Reoxygenation period Myocytes were then reoxygenated with oxygenated Tyrode solution for 30 min at 37°C. The osmolarity of the Tyrode solution was 313 mmol, and all CP were adjusted to an osmolarity of 321 mmol with sucrose. Myocytes were stimulated to contract with 5-ms, 30-V/cm rectangular pulses at a frequency of 1 Hz using an electronic stimulator (SEN-3201, Nihon Kohden, Tokyo, Japan) during the pre-ischemic and reoxygenated periods, in order to acquire diastolic fluorescence value. This was performed only in studies measuring Fluo-3 fluorescence.
Calibration of Fluo-3 fluorescence The Fluo-3 used to measure intracellular Ca2+ concentrations in these experiments makes the comparison of fluorescence ratios impossible, because it does not exhibit spectral shifting after binding Ca2+. Therefore, data are expressed as the change in fluorescence intensity relative to the control fluorescence value. Calibration of Fluo-3 fluorescence intensity was performed by the method described by Li et al. (1987). Briefly, Fluo-3 loaded myocytes, cultured under the same conditions as those used in the experiments, were exposed to a glucose-free buffer containing 3.3 m amytal and 2 m CCCP in order to deplete intracellular ATP so that cells would not contract on exposure to Ca2+. Myocytes were then exposed to various Ca2+ concentrations (40, 150, 350, 600, and 1350 n; calcium calibration buffer kit II) (Hayashi et al., 1994). The [Ca2+]i was equilibrated with the [Ca2+]o by addition of the calcium ionophore ionomycin (10 l). In Fluo-3 loaded cells the relationship between the logarithm of the apparent [Ca2+]i and fluorescence intensity was proportional and was approximated by the equation: −1
[Ca2+]i/[Ca2+]i base=(F/Fbase)(0.997±0.055)
where F is the fluorescence intensity at 530 nm. Fbase and [Ca2+]i base are the diastolic fluorescence intensity and intracellular Ca2+ concentration, respectively, during the pre-ischemic baseline period.
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Statistical analysis
Results Characterization of the experimental model On the second day of culture, when myocytes contracted spontaneously and synchronously, the myocyte beating rate ranged from 110–130 beats/ min at 37°C. On the third day, the number of myocytes increased to 3.0±0.1×105 cells/dish, and the myocyte beating rate increased to 130–150 beats/min. Such cultures were stable for up to 10 days. Myocytes were used on the third day of culture in the current experiments. The cells continued to beat spontaneously after loading with Fluo-3. During the ischemic period, the myocytes immediately stopped contracting after addition of hyperkalemic CP.
Effects of CP Ca2+ and Mg2+ concentrations on [Ca2+]i Figure 1 shows the time course for changes in [Ca2+]i, expressed as changes in fluorescence intensity of Ca2+-bound Fluo-3, during the ischemic and reoxygenation periods. In these studies, the Ca2+ concentration ([Ca2+]o) of the CP was varied, but Mg2+ was not included in the CP. The [Ca2+]i did not change during the first 20 min of ischemia, regardless of [Ca2+]o, but increased to 274±28% (n=46) of the control value in the 0.1 m [Ca2+]o CP group, 155±4% (n=34) in the 0.6 m [Ca2+]o CP group, 577±36% (n=48) in the 1.2 m [Ca2+]o CP group, and 209±23% (n=45) in the 2.4 m [Ca2+]o CP group after 60 min of ischemia. The
2+
[Ca ]i (% of control)
The data from at least 24 myocytes were used to determine [Ca2+]i. For the measurement of the recovery of the myocyte beating rate and the leakage of CPK and LDH, data obtained from six dishes of cultured myocytes were used. All values are expressed as the mean±standard error of the mean (±...). Significant effects were determined using the Kruskal–Wallis non-parametric analysis of variance. If significant differences were found, the multiple comparison procedure was used to identify which groups were significantly different. Moreover, the Pearson’s correlation coefficient was used to analyse correlations between [Ca2+]i, the beating rate recovery, and the leakage of CPK and LDH. Differences were considered significant if P<0.05.
Simulated ischemia (NaCN, glucose-free)
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500 400 300 200 100 0
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40 50 60 Time (min)
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Figure 1 Effects of the Ca2+ concentration in Mg2+-free CP on intracellular Ca2+ concentration ([Ca2+]i). Data shown represent the mean±... from 34 to 48 myocytes per group. (Χ), 0.1 m; (Ε), 0.6 m; (Η), 1.2 m; (Β), 2.4 m. (∗ P<0.01, comparing [Ca2+]i after 60 min of ischemia for 1.2 m group to [Ca2+]i for the other groups; † P<0.01, comparing the maximum [Ca2+]i during reoxygenation for 0.1 m group to [Ca2+]i for the other groups).
[Ca2+]i in the 1.2 m [Ca2+]o CP group was significantly greater than the other CP groups following 60 min (P<0.01). The [Ca2+]i initially increased in all groups during the subsequent reoxygenation period. The maximum [Ca2+]i during reoxygenation were 771±147, 166±5, 622±38, and 250±30% in the 0.1, 0.6, 1.2, and 2.4 m [Ca2+]o CP groups, respectively. The [Ca2+]i during reoxygenation in the 0.1 m [Ca2+]o group significantly increased to a greater extent compared to the other groups (P<0.01). For each [Ca2+]o, the [Ca2+]i after 60 min of ischemia and the maximum [Ca2+]i during reoxygenation varied with the Mg2+ concentration ([Mg2+]o) in the CP (Fig. 2). In 0.1 m [Ca2+]o CP, [Ca2+]i after 60 min of ischemia was significantly lower for CP containing 1.2 m Mg2+, 8 m Mg2+, and 16 m Mg2+ than CP free of Mg2+ (P<0.01). After perfusion with 0.1 m [Ca2+]o CP, the maximum [Ca2+]i during reoxygenation was also significantly lower for CP containing 1.2 m Mg2+ (115±18%), 8 m Mg2+ (166±13%), and 16 m Mg2+ (184±17%) than CP free of Mg2+ (P<0.01). However, the maximum [Ca2+]i during reoxygenation increased to a greater extent if the [Mg2+]o was greater than 1.2 m in the CP. In the 0.6 m [Ca2+]o CP group, [Ca2+]i after 60 min of ischemia at a [Mg2+]o of 1.2, 8, and 16 m were 134±14, 97±4, and 101±2%, respectively. [Ca2+]i after 60 min of ischemia at 8 m and 16 m [Mg2+]o were significantly lower than in cells perfused with CP without Mg2+ (P<0.01). The maximum [Ca2+]i during reoxygenation at a
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Calcium and Magnesium in Cardioplegia for Immature Hearts 200
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[Ca2+]o = 0.6 mM
2+ [Ca ]o = 0.1 mM
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[Ca2+]i (% of control)
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Figure 2 Relationship between intracellular Ca2+ concentration ([Ca2+]i) and Mg2+ concentration ([Mg2+]o) for each Ca2+ concentration ([Ca2+]o) CP. Closed histograms represent [Ca2+]i after 60 min of ischemia. The open histograms represent the maximum [Ca2+]i during reoxygenation. Data shown represent the mean±... from 24 to 48 myocytes per group. (∗ P<0.01, compared to [Ca2+]i after 60 min of ischemia when no Mg2+ was present; † P<0.01, compared to the maximum [Ca2+]i during reoxygenation when no Mg2+ was present; ‡ P<0.05 and § P<0.01 compared to the maximum [Ca2+]i during reoxygenation for 1.2 m [Mg2+]o).
[Mg2+]o of 1.2 m, 8 m, and 16 m were 148±16, 101±5, and 107±2%, respectively. The maximum [Ca2+]i during reoxygenation in the presence of 8 or 16 m Mg2+ was also significantly lower than if Mg2+ was absent (P<0.01). In the 1.2 m [Ca2+]o CP group, [Ca2+]i after 60 min of ischemia at 1.2 m [Mg2+]o, 8 m [Mg2+]o, and 16 m [Mg2+]o were significantly less than values obtained for CP without Mg2+ (P<0.01). The maximum [Ca2+]i during reoxygenation at 1.2 m [Mg2+]o, 8 m [Mg2+]o, and 16 m [Mg2+]o were also significantly lower than values for CP without Mg2+ (P<0.01). In the 2.4 m [Ca2+]o CP group, [Ca2+]i after 60 min of ischemia at a [Mg2+]o of 1.2, 8, and 16 m were 161±12, 144±24, and 118±17%, respectively. [Ca2+]i after 60 min of ischemia at 16 m [Mg2+]o was significantly lower than if CP contained no Mg2+ (P<0.01). The maximum [Ca2+]i during reoxygenation at a [Mg2+]o of 1.2, 8, and 16 m were 166±14, 155±26, and 104±12%, respectively. The maximum [Ca2+]i reoxygenation at 16 m [Mg2+]o CP was significantly less than if CP contained no Mg2+ (P<0.01).
Cardiac myocyte beating recovery after ischemia In the 0.1 m [Ca2+]o CP group, the beating rate recovery at 1.2, 8, and 16 m [Mg2+]o were better than CP without Mg2+ (P<0.01). In this group, there was no recovery of beating because hypercontracture had developed early during reoxygenation if there was no Mg2+ in the CP. In the 1.2 m [Ca2+]o CP group, the beating rate recovery at 8 and 16 m [Mg2+]o were significantly better than CP without Mg2+ (P<0.05). In the 0.6 and 2.4 m [Ca2+]o CP groups, in which the increased [Ca2+]i during ischemia and reoxygenation in Mg2+-free CP were minute, the beating rate recovery was not significant irrespective of the presence of extracellular Mg2+ (Table 1). In addition, with the exception of 1.2 m [Ca2+]o CP groups, beating rate recovery indicated a [Ca2+]o dependent increase at 0 m [Mg2+]o. There was a significant correlation between the maximum [Ca2+]i through the ischemia and reoxygenation periods and the beating rate recovery for all of the groups (r=−0.74, P<0.001).
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T. Ichiba et al. Table 1 Dose responses to Ca2+ and Mg2+ concentrations in CP Ca2+ and Mg2+ concentrations (m) Ca2+ 0.1 Mg2+0 1.2 8 16 P value Ca2+ 0.6 Mg2+0 1.2 8 16 P value Ca2+ 1.2 Mg2+0 1.2 8 16 P value Ca2+ 2.4 Mg2+0 1.2 8 16 P value
Beating rate recovery (%)
CPK leakage (mIU/dish)
LDH leakage (mIU/dish)
0 61±7∗ 54±5∗ 47±13∗ <0.01
23.6±1.0 15.0±0.3∗ 15.6±0.2∗ 17.0±0.6∗ <0.01
27.9±1.2 7.9±0.4∗ 8.6±0.4∗ 9.3±1.0∗ <0.01
80±2 89±8 90±6 85±18 ..
16.5±0.3 14.4±0.7 13.4±0.7 14.6±0.8 ..
11.8±0.2 9.2±0.6∗ 8.1±0.4∗ 8.8±0.5∗ <0.01
55±6 71±7 75±4† 80±15† <0.05
20.1±1.4 17.4±0.5 16.4±1.0† 15.7±0.6† <0.05
12.7±0.1 9.1±0.9∗ 8.8±0.4∗ 9.1±0.3∗ <0.01
97±10 98±2 95±3 97±17 ..
16.0±0.1 14.9±0.1 15.1±0.6 14.9±0.3 ..
8.8±0.2 8.8±0.6 7.9±0.4 8.2±1.1 ..
The beating rate recovery after 30 min of reoxygenation is expressed as a percentage of the beating rate before ischemia. CPK and LDH leakages represent the total quantity during both ischemia and reoxygenation. Values are expressed as mean±..., with n=6 dishes in each group. ∗ P<0.01, † P<0.05, compared to 0 m Mg2+ CP group for each Ca2+ concentration. ..=not significant (P[0.05).
Enzyme leakage from myocytes during ischemia and reoxygenation Table 1 shows the total amount of CPK and LDH released from myocytes during both the ischemia and reoxygenation periods for each group. In the 0.1 m [Ca2+]o CP group, the CPK and LDH leakages at a [Mg2+]o of 1.2, 8, and 16 m were significantly lower than at 0 m [Mg2+]o. In the 0.6 m [Ca2+]o CP group, the release of LDH at a [Mg2+]o of 1.2, 8, and 16 m were significantly lower than at 0 m [Mg2+]o. However, the release of CPK did not differ significantly in this group. In the 1.2 m [Ca2+]o CP group, CPK leakage at a [Mg2+]o of 8 and 16 m and LDH release at 1.2, 8 and 16 m [Mg2+]o were significantly lower than at 0 m [Mg2+]o. In the 2.4 m [Ca2+]o CP group, neither CPK nor LDH leakage were affected by the [Mg2+]o. The enzyme leakages were also significantly lower in the 0.6 and 2.4 m [Ca2+]o CP groups than in the 0.1 and 1.2 m [Ca2+]o CP groups when the CP contained no Mg2+ (not shown in Table 1). Ultimately, the leakage of CPK and
LDH was significantly less in groups in which the increase in [Ca2+]i during both ischemia and reoxygenation were prevented (the correlation between the maximum [Ca2+]i through the ischemia and reoxygenation periods and CPK: r=0.945, P<0.0001; between the maximum [Ca2+]i and LDH: r=0.862, P<0.0001).
The correlation between beating rate recovery and enzyme leakage There was a significant correlation noted between the beating rate recovery and the leakage of CPK (r=−0.835, P<0.0001) and LDH (r=−0.794, P<0.0001). In comparison in each [Ca2+]o CP group response to [Mg2+]o, the beating rate recovery correlated significantly with the leakage of CPK in the 0.1 and 1.2 m [Ca2+]o CP groups and the leakage of LDH in 0.1 m [Ca2+]o CP group. On the other hand, in comparison in each [Mg2+]o, there was a significant correlation between the beating rate
Calcium and Magnesium in Cardioplegia for Immature Hearts
recovery and the enzyme leakage (both CPK and LDH) only in 0 m [Mg2+]o.
Discussion Our investigation demonstrates that the [Ca2+]o in hyperkalemic CP influences [Ca2+]i and myocardial protection in immature cardiac myocytes. The effect of extracellular Ca2+ was significant, especially when there was no Mg2+ present in the CP. Further, an optimized [Mg2+]o in CP inhibited increases in [Ca2+]i and attenuated cell damage during ischemia and reoxygenation. However, a high [Mg2+]o relative to a low [Ca2+]o in CP, such as in the 0.1 m [Ca2+]o CP group, resulted in Ca2+ overload during reoxygenation. Therefore, a high [Mg2+]o may be detrimental if [Ca2+]o in CP is very low. In contrast, the effects of Mg2+ on myocardial protection may be minimal, if [Ca2+]o in CP is as high as 2.4 m.
Characteristics of Ca2+ handling in immature myocytes Calcium overload, resulting from the failure of intracellular Ca2+ homeostasis during ischemia and reperfusion, is one of the main causes of ischemic myocardial injury. Several potential mechanisms may be responsible for alterations in [Ca2+]i: (1) changes in Na+/Ca2+ exchange; (2) alterations in sarcolemmal voltage-gated Ca2+ channels; and (3) changes in the Ca2+ release channel in the sarcoplasmic reticulum (SR). Recent studies have demonstrated that immature myocytes have a poorly developed SR, and as a result are more dependent on Ca2+ influx across the sarcolemma to generate tension (Anderson, 1989; Chin et al., 1990; Kaufman et al., 1990). Moreover, Huynh et al. (1992) and Wetzel et al. (1993) have shown that voltagegated Ca2+ channels are relatively deficient in immature myocytes. To compensate, Na+/Ca2+ exchange increases in immature myocytes (Artman, 1992). Accordingly, it is believed that in immature myocytes, sarcolemmal Ca2+ influx pathways, especially the Na+/Ca2+ exchanger, are important in the control of [Ca2+]i during ischemia and reperfusion.
The effect of Ca2+ in CP on [Ca2+]i during ischemia and reoxygenation The results of this study show that alterations in the Ca2+ concentration of CP without Mg2+ result in various patterns of intracellular Ca2+ overload during ischemia and reoxygenation in immature
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myocytes. This suggests the existence of several mechanisms that may be responsible for Ca2+ overload. Na+/Ca2+ exchanger activity, which is important for [Ca2+]i handling in the ischemic immature myocyte, is dependent on the concentration gradients of both Na+ and Ca2+ across the sarcolemma. In addition, the exchanger is dependent on membrane potential because of the electrogenicity of the exchange (3:1 Na+/Ca2+ exchange). This is based on studies that have shown that Ca2+ influx via reversal of the exchanger is enhanced in depolarized membranes (Wetzel et al., 1995). As anaerobic metabolism increases in the myocyte during ischemia, protons (H+) accumulate in the cytoplasm. If the extracellular pH does not become acidic because of perfusion with CP, the activity of the Na+/ H+ exchanger is not inhibited during ischemia. As a result, H+ accumulating in myocytes during ischemia are exchanged with extracellular Na+. The Na+ which enters the cell via the Na+/H+ exchanger is subsequently exchanged with extracellular Ca2+ via the Na+/Ca2+ exchanger. In addition, reversal of the Na+/Ca2+ exchanger results in faster exchange because the membrane potential becomes relatively more depolarized by hyperkalemic CP during ischemia. Taken together, these changes result in intracellular Ca2+ overload. The glycocalyx on the sarcolemma plays an important role in determining the [Ca2+]o and membrane potential by binding extracellular Ca2+. The glycocalyx can therefore regulate the activity of the Na+/Ca2+ exchanger (Langer, 1984). As a result, [Ca2+]i during ischemia and reoxygenation may be regulated by the degree of binding of Ca2+ to the glycocalyx. In our experiments, the increases in [Ca2+]i during ischemia and reoxygenation were suppressed to a greater extent in myocytes perfused with 0.6 or 2.4 m [Ca2+]o CP compared to myocytes perfused with 0.1 or 1.2 m [Ca2+]o CP when Mg2+ was not present. We suggest that the following mechanisms are responsible for this effect. First, in 0.6 m [Ca2+]o without Mg2+ CP, the amount of Ca2+ binding to the glycocalyx does not reverse the Na+/ Ca2+ exchanger either during ischemia or during reoxygenation with Tyrode solution containing 1 m [Ca2+]o and 1 m [Mg2+]o. In 1.2 m [Ca2+]o CP, severe overload occurs during ischemia because the increased binding of Ca2+ to the glycocalyx reverses Na+/Ca2+ exchange during ischemia. Finally, in 2.4 m [Ca2+]o CP, although the amount of Ca2+ bound to the glycocalyx increases, the membrane potential is greater than in 1.2 m [Ca2+]o because of the presence of excess Ca2+. As
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a result, the degree of depolarization caused by hyperkalemic CP decreases during the ischemic period. This effect may decrease the reversed activity of the Na+/Ca2+ exchanger, resulting in attenuated increases in [Ca2+]i. In 0.1 m [Ca2+]o CP, severe Ca2+ overload occurred during reoxygenation, suggesting the existence of a mechanism different from those described above. When myocytes were perfused with a low [Ca2+]o CP, such as 0.1 m, the glycocalyx-sarcolemma complex may have been partially disrupted. This disruption of a Ca2+ “trap” significantly changes the free [Ca2+]o at the sarcolemma. Accordingly, myocytes perfused with 0.1 m [Ca2+]o CP experience increased Ca2+ influx during subsequent reperfusion with 1 m [Ca2+]o, because the reversal of Na+/Ca2+ exchanger activity is augmented by the rapid increase in [Ca2+]o at the sarcolemma during reperfusion (i.e. “calcium paradox”) (Zimmerman and Hulsmann, 1966). The [Ca2+]o of CP therefore affects several important factors: (1) the stability of the glycocalyx-sarcolemmal complex; (2) the amount of Ca2+ binding to the glycocalyx, which in turn determines the quantity of Ca2+ that can enter cells; and (3) the membrane potential which determines both the polarity and the activity of the Na+/Ca2+ exchanger.
The effect of Mg2+ on [Ca2+]i during ischemia and reoxygenation In the adult heart, CP that contain Mg2+ produce better post-ischemic ventricular function than those that do not contain Mg2+ (Hearse et al., 1978; Reynolds et al., 1989). The exact protective mechanism of Mg2+ is not known at this time, although several mechanisms have been suggested. The first is that Mg2+ acts as a cofactor for energy-requiring intracellular reactions. Specifically, Mg2+ regulates both cyclic AMP-dependent voltage-gated Ca2+ channels and Ca2+ release from the SR (Agus and Morad, 1991). Furthermore, intracellular Mg2+ is important for Na+/K+ ATPase activity, which promotes the active transport of Na+ out of the cell. The resulting decrease in intracellular Na+ suppresses Ca2+ influx by reversing the Na+/Ca2+ exchanger. However, the cell membrane has a relatively low permeability to Mg2+ (Flatman, 1984), and the concentration of intracellular Mg2+ is unlikely to decrease significantly during ischemia or hypoxia (Fry, 1986; Murphy et al., 1989). A second mechanism involves the ability of Mg2+ to act as a physiologic inhibitor of Ca2+
transport across the sarcolemma (Iseri and French, 1984). In our study, the addition of Mg2+ to CP significantly suppressed intracellular Ca2+ overload as well as the beating rate recovery and/or enzyme leakage during ischemia and reoxygenation, with the exception of the 2.4 m [Ca2+]o CP group. However, the addition of more than 8 m Mg2+ to 0.1 m [Ca2+]o CP resulted in an increase in [Ca2+]i during reoxygenation, and poorer myocardial protection. The protective effects of Mg2+ cannot be ascribed solely to Ca2+ channel inhibition, especially in immature myocytes, in which Ca2+ channels are relatively deficient. Langer (1984) has suggested that Mg2+ competes with Ca2+ for binding sites within the sarcolemmal–glycocalyx complex of the myocyte. As a result, the addition of Mg2+ to CP decreases the magnitude of the Ca2+ influx via Ca2+ channels or the Na+/Ca2+ exchanger during ischemia because of a decrease in the relative [Ca2+]o. Moreover, the Mg2+ in CP prevents severe Ca2+ overload during reoxygenation after perfusion with low [Ca2+]o CP (i.e. “calcium paradox”) by preserving the sarcolemmal–glycocalyx complex. In contrast, a high [Mg2+]o relative to a low [Ca2+]o increases the Mg2+ binding of the glycocalyx, and inhibits Ca2+ influx into the cell during ischemia. However, Mg2+ is the least effective of the divalent cations in displacing Ca2+ from binding sites (Langer, 1984). Therefore, during reoxygenation, the Mg2+ bound to the glycocalyx is readily displaced by Ca2+. This would increase the [Ca2+]o rapidly and increase the exchange of intracellular Na+ for Ca2+ during reoxygenation. In 0.6 m [Ca2+]o CP, a [Mg2+]o greater than 8 m did not suppress the [Ca2+]i increase. It may be that the addition of Mg2+ at concentrations greater than 16 m to CP causes increases in [Ca2+]i during reoxygenation. Accordingly, we hypothesize that [Mg2+]o affects the [Ca2+]i less in a high [Ca2+]o CP group such as 2.4 m than the other [Ca2+]o CP groups.
The relationship between [Ca2+]o and cardioprotection In our study, the recovery of the beating rate and the release of CPK and LDH were used as parameters of the cardioprotective effect of CP. The recovery of the beating rate improved, and the release of both CPK and LDH were less in groups in which the increases in [Ca2+]i were suppressed during ischemia and reoxygenation. We found a statistically significant correlation between [Ca2+]i and either
Calcium and Magnesium in Cardioplegia for Immature Hearts
beating rate recovery or enzyme release. This suggests that there is a close correlation between the cardioprotective effects of CP and the regulation of [Ca2+]i during ischemia and reoxygenation. When CP contained no Mg2+, especially in the 0.1 m [Ca2+]o CP group, the damage would be due to rapid, severe intracellular Ca2+ overload resulting in cell destruction by hypercontracture during reoxygenation. When [Mg2+]o was varied in each [Ca2+]o CP group, the recovery of beating rate improved and the release of CPK and LDH were less in groups in which the increases in [Ca2+]i were suppressed during ischemia and reoxygenation. The beating rate recovery and enzyme leakage were not dependent on [Mg2+]o in the 2.4 m [Ca2+]o CP group. We hypothesize the reason that in the 2.4 m [Ca2+]o CP group the suppressing effect of Mg2+ on the [Ca2+]i increase may have been less than in the other [Ca2+]o CP groups. On the other hand, in comparison in each [Mg2+]o, there was no significant correlation between the beating rate recovery and the enzyme leakage, with exception of 0 m [Mg2+]o. The reason is considered to be that for 1.2, 8 and 16 m [Mg2+]o CP groups, the [Ca2+]i varied with [Ca2+]o in CP less compared with 0 m [Mg2+]o CP group. Consequently, in those groups, the beating rate recovery and the enzyme leakage did not vary so significantly as this group statistically.
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similar to that in ischemia. Tyrode solution containing 1 m Ca2+ and 1 m Mg2+ was perfused during the reoxygenation period in all experiments, in spite of using the CP which have various concentrations of Ca2+ and Mg2+ during ischemia, because we were attempting to simulate the clinical situation; namely, reperfusion by blood containing about 1 m Ca2+ and 1 m Mg2+ after cardiac arrest.
Conclusion The current study demonstrates that both Ca2+ and Mg2+ in CP influence cardioprotective effects by regulating the intracellular Ca2+ concentration during ischemia and reoxygenation in immature myocytes. Mg2+ in CP suppressed increases in the [Ca2+]i during ischemia and subsequent reoxygenation, thereby conferring protection. However, too great a [Mg2+]o relative to low [Ca2+]o may cause cell injury through Ca2+ overload during reoxygenation, while the protective effects of [Mg2+]o are weak if CP contains high [Ca2+]o. These results indicate that these cations in CP determine the amount of Ca2+ that enters the cell via the sarcolemma. Further, this influx is determined by both Na+/Ca2+ exchange and the glycocalyx in immature myocytes. This study helps to elucidate the cardioplegic effect of Ca2+ and Mg2+ in CP for immature hearts.
Methodologic considerations
References In the present study, we used neonatal myocyte monolayers on the third day of culture as a model of immature myocardium. A well-defined myocyte culture system is useful in evaluating the direct effects of various stimuli, including ischemia or post-ischemic reperfusion (Orita et al., 1994), because the myocytes are not affected by other cell components such as endothelial cells or fibroblasts. Moreover, a monolayer of cells maintains a constant synchronous beating rate. We simulated ischemia using a glucose-free perfusion with sodium cyanide. Depletion of intracellular ATP was achieved by metabolic inhibition with the perfusion. Although intracellular ATP was not measured in the present study, other investigators have reported that ATP is reduced by approximately 50% in neonatal cardiomyocytes after 90 min of either sodium cyanide induced metabolic stress or true hypoxia (Iwaki et al., 1993). The previous data indicate that the phenomenon which occurs in the present study is
A ZS, M M, 1991. Modulation of cardiac ion channels by magnesium. Annu Rev Physiol 53: 299– 307. A PAW, 1989. Maturation and cardiac contractility. Cardiol Clin 7: 209–225. A M, 1992. Sarcolemmal Na–Ca exchange activity and exchanger immunoreactivity in developing rabbit hearts. Am J Physiol 263: H1506–H1513. A K, C D, MC J, L S, F H, 1993. Magnesium cardioplegia prevents accumulation of cytosolic calcium in the ischemic myocardium. J Mol Cell Cardiol 25: 1387–1390. B PS J, H FW, P GL, C RE, 1991. Magnesium ion is beneficial in hypothermic crystalloid cardioplegia. Ann Thorac Surg 51: 359–367. C TK, F WF, K TS, 1990. Developmental changes in cardiac myocyte calcium regulation. Circ Res 67: 574–579. F PW, 1984. Magnesium transport across cell membranes. J Membr Biol 80: 1–14. F CH, 1986. Measurement and control of intracellular magnesium ion concentration in guinea pig and ferret ventricular myocardium. Magnesium 5: 306–331. H H, S H, N N, T H, H M,
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Y Y, K A, Y N, 1994. Simultaneous measurement of intracellular Na+ and Ca2+ during K+-free perfusion in isolated myocytes. Am J Physiol 266: C416–C422. H DJ, S DA, B MV, 1978. Myocardial protection during ischemic cardiac arrest: the importance of magnesium in cardioplegic infusates. J Thorac Cardiovasc Surg 75: 877–885. H J, M F, V G, 1981. Perinatal growth of the rabbit cardiac cell: possible implications for the mechanisms of relaxation. J Mol Cell Cardiol 13: 725–740. H TV, C F, W GT, F WF, K TS, 1992. Developmental changes in membrane Ca2+ and K+ currents in fetal, neonatal, and adult heart cells. Circ Res 70: 508–515. I LT, F JH, 1984. Magnesium: nature’s physiologic calcium blocker. Am Heart J 108: 188–193. I K, C SH, D WH, M R, 1993. Induction of hsp70 in cultured rat neonatal cardiomyocytes by hypoxia and metabolic stress. Circulation 87: 2023–2032. K TM, H JW, W DJ, M L, 1990. Age-related changes in myocardial relaxation and sarcoplasmic reticulum function. Am J Physiol 259: H309– H346. L GA, 1984. Calcium at the sarcolemma. J Mol Cell Cardiol 16: 147–153. L Q, A RA, S BT, 1987. Quantitation of intracellular free calcium in single adult cardiomyocytes by fura-2 fluorescence microscopy: calibration of fura-2 ratios. Biochem Biophys Res Common 147: 120–126. ML KT, 1989. Effects of hypoxia and metabolic inhibition on the intracellular sodium activity of mammalian ventricular muscle. J Physiol 416: 455–468. M JG, 1982. Excitation–contraction coupling in neonatal and adult myocardium of cat. Am J Physiol 242: H834–H843.
M E, S C, L LA, R B, L RE, 1989. Cytosolic free magnesium levels in ischemic rat heart. J Biol Chem 264: 5622–5627. N T, J JM, 1984. Developmental changes in myocardial mechanical function and subcellular organelles. Am J Physiol 246: H615–H625. N WG, E JS, 1986. Reperfusion injury: laboratory artifact or clinical dilemma? Circulation 74: 215–221. O H, F M, H S, U H, FK K, K M, W M, 1994. Cardiac myocyte functional and biochemical changes after hypothermic preservation in vitro. J Thorac Cardiovasc Surg 107: 226– 232. O H, P MR, J RM, D DK, 1989. Mechanism of membrane phospholipid degradation in ischemic-reperfused rat hearts. Am J Physiol 257: H252–H258. R TR, G GA, T JS, O’K DD, D WM, 1989. Myocardial preservation related to magnesium content of hyperkalemic cardioplegic solutions at 8°C. Ann Thorac Surg 47: 907–913. S P, S S, 1982. Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells. Circ Res 50: 101–116. T N, K H, H T, H Y, I S, M T, 1994. Effect of magnesium and calcium on myocardial protection by cardioplegic solutions. Ann Thorac Surg 57: 177–182. W GT, C F, K TS, 1993. Ca2+ channel kinetics in acutely isolated fetal neonatal and adult rabbit cardiac myocytes. Circ Res 72: 1065–1074. W GT, C F, K TS, 1995. Na+/Ca2+ exchange and cell contraction in isolated neonatal and adult rabbit cardiac myocytes. Am J Physiol 268: H1723–H1733. Z ANE, H WC, 1966. Paradoxical influence of calcium ions on the permeability of the cell membranes of the isolated rat heart. Nature 211: 646–647.
Regulation of Intracellular Calcium Concentrations by Calcium and Magnesium in Cardioplegic Solutions Protects Rat Neonatal Myocytes from Simulated Ischemia Takashi Ichiba, Naruto Matsuda, Naoaki Takemoto, Shingo Ishiguro, Hiroaki Kuroda and Tohru Mori Department of Surgery, Tottori University, Faculty of Medicine, Yonago, Japan (Received 19 November 1997, accepted in revised form 23 February 1998) T. I, N. M, N. T, S. I, H. K T. M. Regulation of Intracellular Calcium Concentrations by Calcium and Magnesium in Cardioplegic Solutions Protects Rat Neonatal Myocytes from Simulated Ischemia. Journal of Molecular and Cellular Cardiology (1998) 30, 1105–1114. The effects of calcium and magnesium ions in cardioplegic solutions on cardioprotection and intracellular calcium ion handling during ischemia and reoxygenation were investigated in cultured neonatal rat myocardial cells. Myocytes were subjected to simulated ischemia for 60 min at 37°C in hyperkalemic cardioplegic solutions containing various concentrations of calcium and magnesium ions, followed by 30 min of reoxygenation. For each Ca2+ concentration (0.1, 0.6, 1.2, or 2.4 m), the Mg2+ concentration was either 0, 1.2, 8, or 16 m. The increase in intracellular Ca2+ concentration during ischemia and reoxygenation was suppressed by the addition of magnesium ion, independent of cardioplegic Ca2+ concentration. The recovery of spontaneous contraction rate and enzyme leakage (creatine phosphokinase and lactate dehydrogenase) during both ischemia and reoxygenation correlated with the degree of inhibition of intracellular Ca2+ accumulation. However, in the 0.1 m Ca2+ groups in which the Mg2+ concentration was greater than 8 m, the intracellular Ca2+ concentration increased during reoxygenation in a dose-dependent fashion of Mg2+, and was associated with increased enzyme leakage. The findings suggest that in immature cardiac myocytes, the concentrations of Ca2+ and Mg2+ present in cardioplegic solutions control the intracellular Ca2+ concentration during ischemia and reoxygenation, which, in turn, influences the cardioprotective effect of the cardioplegic solution. 1998 Academic Press K W: Cardioplegic solution; Simulated ischemia; Intracellular calcium; Fluo-3; Cell injury; Immature myocytes; Calcium ion; Magnesium ion.
Introduction Abnormal handling of intracellular calcium ions (Ca2+) during ischemia and reperfusion is one of the principal causes of post-ischemic myocardial dysfunction. Increased intracellular Ca2+ stimulates a number of calcium-dependent enzymes, including phospholipases and proteases, which are potentially
harmful to the cell (Nayler and Elz, 1986; Otani et al., 1989). The use of hyperkalemic cardioplegic solutions (CP) attenuates the increase in the intracellular Ca2+ concentration ([Ca2+]i) that occurs during ischemia and improves post-ischemic contractile function (Ataka et al., 1993). The individual components of CP have been studied in an attempt to optimize the inhibition of intracellular Ca2+
Please address all correspondence to: Takashi Ichiba, Department of Surgery, Tottori University Faculty of Medicine, 36-1 Nishi-machi, Yonago, Tottori 683, Japan.
0022–2828/98/061105+10 $30.00/0
1998 Academic Press
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accumulation and thereby improve myocardial protection. The concentrations of Ca2+ and magnesium ions (Mg2+) in cardioplegic solutions have been found to be important in myocardial protection (Brown et al., 1991; Ataka et al., 1993; Takemoto et al., 1994). Specifically, the extracellular Ca2+ concentration ([Ca2+]o) alters the amount of Ca2+ bound to the sarcolemma, thus determining the magnitude of Ca2+ influx (Langer, 1984). Further, Mg2+ acts as a physiologic Ca2+ channel blocker (Iseri and French, 1984), decreasing Ca2+ influx into the cell. Previous studies of Ca2+ and Mg2+ metabolism have not adequately examined the responses of the immature heart. In recent studies, it has been shown that Ca2+ handling in immature myocardial cells differs greatly from that in the mature myocyte. The difference arises from the fact that an immature myocyte possesses little sarcoplasmic reticulum (SR), and regulates intracellular Ca2+ predominantly at the level of the sarcolemma. In contrast, a mature myocyte relies largely on its developed SR to control intracellular Ca2+ (Hoerter et al., 1981; Maylie, 1982; Nakanishi and Jarmakani, 1984). Therefore, the effects of extracellular Ca2+ and Mg2+, which control Ca2+ influx via the sarcolemma, may be more important for immature hearts than mature hearts. In the present report, neonatal, cultured myocardial cells were used to determine the effects of Ca2+ and Mg2+ in hyperkalemic CP on the regulation of intracellular Ca2+ and myocardial protection during both ischemia and reoxygenation.
Cell culture Cultured neonatal rat myocardial cells were prepared by a modification of the method used by Simpson and Savion (1982). In brief, ventricles from 2- to 3-day-old Wistar rats were minced and dissociated at 37°C using 0.1% collagenase. The digestion medium was discarded, and the cells were resuspended in culture medium (M199), supplemented with 4% FBS, 100 U/ml penicillin, 100 lg/ml streptomycin, 10 lg/ml transferrin, and 10 lg/ml insulin. The cell suspension was preplated on a 60-mm diameter plastic dish for 30 min at 37°C in a CO2 incubator. The non-attached, viable cells were seeded onto type I collagen-coated 35mm dishes (2×105 cells per dish) and incubated for 3 days prior to use.
Cell loading with Fluo-3 Fluo-3 AM was used to determine [Ca2+]i. Myocardial cells were incubated in M199 containing 10 l Fluo-3 AM for 30 min at 37°C. Extracellular Fluo-3 AM was then removed by changing the medium twice with M199. This loading period was followed by a 20-min post-incubation period at 37°C, which allowed complete hydrolysis of the Fluo-3 AM ester.
[Ca2+]i measurements
Materials and Methods Materials Reagents used in this study included: collagenase, transferrin (Wako Chemical, Osaka, Japan), medium 199 (M199), penicillin, streptomycin (BioWhittaker, Walkersville, MD, USA), insulin (Becton Dickinson, Two Oak Park, MA, USA), fetal bovine serum (FBS) (Cansera, Ontario, Canada), the acetoxymethyl ester Fluo-3 (Fluo-3 AM) (Dojindo, Kumamoto, Japan), amytal, carbonyl cyanide mchlorophenylhydrazone (CCCP) (Sigma, St Louis, MO, USA), ionomycin (Calbiochem-Novabiochem, La Jolla, CA, USA), and calcium calibration buffer kit II (Molecular Probes, Eugene, OR, USA). Rats were treated in accordance with the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals.
We employed a confocal laser scanning system (INSIGHT PLUS, Meridian Instruments, Okemos, MI, USA) equipped with an Olympus IMT-2 inverted microscope to measure [Ca2+]i. Intracellular Fluo3 was excited at 488 nm, and its emission fluorescence was measured at 530 nm. The fluorescence images were recorded using a CCD camera equipped with an intensifier and stored on video tape (30 frames per s). In order to minimize photobleaching, the fluorescence acquisition was performed for 3 s at 5-min intervals. In between data acquisition, the cells were not exposed to the exciting wavelength light. The data were analysed using INSIGHT-IQ software. The diastolic fluorescence values during a 3-s period were averaged for individual cells. In preliminary experiments, the fluorescence intensity of Fluo-3 did not decrease significantly after 120 min (data not shown).
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Beating rate recovery measurements The beating rate of synchronized myocytes was determined by viewing the cultured cells microscopically. The beating rate per min was recorded prior to ischemic period and after 30 min of reoxygenation that followed the ischemic period. The recovery ratio for the beating rate during reoxygenation is expressed as a percentage of the beating rate prior to ischemic period.
Enzyme leakage determination The media used during ischemic and reoxygenated period were assayed spectrophotometrically to determine the quantity of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) released from cells. The enzyme activities were determined using the uv-rate and Wro´blewski–La Due methods employing a uv-visible recording spectrophotometer (UV-160, Simadzu, Kyoto, Japan). The total amounts of CPK and LDH released were expressed as international milli units (mIU) per dish.
Experimental protocol
Pre-ischemic period Cultured myocardial cells were placed in a temperature-controlled (37°C) chamber mounted on the stage of an inverted microscope. A pair of platinum stimulating electrodes were placed 2-cm apart in the chamber. The myocytes were first incubated for 30 min with Tyrode solution containing: 140 m NaCl, 6 m KCl, 1 m CaCl2, 10 m glucose, and 5 m N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) (Macleod, 1989). The medium was equilibrated with 100% O2 at 37°C, and the pH was adjusted to 7.4 with NaOH. The beating rate and fluorescence of Fluo-3 bound to intracellular Ca2+ were recorded as control values during the last 1 min of this period.
Ischemic period Myocytes were then subjected to chemically-induced hypoxic, glucose-free perfusion simulating ischemia for 60 min at 37°C. The solutions (cardioplegic solutions; CP) contained: 110 m NaCl, 16 m KCl, 5 m HEPES, 2 m sodium cyanide to inhibit oxidative phosphorylation, and varying concentrations of CaCl2 and MgCl2. The pH of these
solutions were adjusted to 7.4 with NaOH. The [Ca2+]o was either 0.1, 0.6, 1.2, or 2.4 m. For each [Ca2+]o, the Mg2+ concentration ([Mg2+]o) was 0, 1.2, 8, or 16 m.
Reoxygenation period Myocytes were then reoxygenated with oxygenated Tyrode solution for 30 min at 37°C. The osmolarity of the Tyrode solution was 313 mmol, and all CP were adjusted to an osmolarity of 321 mmol with sucrose. Myocytes were stimulated to contract with 5-ms, 30-V/cm rectangular pulses at a frequency of 1 Hz using an electronic stimulator (SEN-3201, Nihon Kohden, Tokyo, Japan) during the pre-ischemic and reoxygenated periods, in order to acquire diastolic fluorescence value. This was performed only in studies measuring Fluo-3 fluorescence.
Calibration of Fluo-3 fluorescence The Fluo-3 used to measure intracellular Ca2+ concentrations in these experiments makes the comparison of fluorescence ratios impossible, because it does not exhibit spectral shifting after binding Ca2+. Therefore, data are expressed as the change in fluorescence intensity relative to the control fluorescence value. Calibration of Fluo-3 fluorescence intensity was performed by the method described by Li et al. (1987). Briefly, Fluo-3 loaded myocytes, cultured under the same conditions as those used in the experiments, were exposed to a glucose-free buffer containing 3.3 m amytal and 2 m CCCP in order to deplete intracellular ATP so that cells would not contract on exposure to Ca2+. Myocytes were then exposed to various Ca2+ concentrations (40, 150, 350, 600, and 1350 n; calcium calibration buffer kit II) (Hayashi et al., 1994). The [Ca2+]i was equilibrated with the [Ca2+]o by addition of the calcium ionophore ionomycin (10 l). In Fluo-3 loaded cells the relationship between the logarithm of the apparent [Ca2+]i and fluorescence intensity was proportional and was approximated by the equation: −1
[Ca2+]i/[Ca2+]i base=(F/Fbase)(0.997±0.055)
where F is the fluorescence intensity at 530 nm. Fbase and [Ca2+]i base are the diastolic fluorescence intensity and intracellular Ca2+ concentration, respectively, during the pre-ischemic baseline period.
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Statistical analysis
Results Characterization of the experimental model On the second day of culture, when myocytes contracted spontaneously and synchronously, the myocyte beating rate ranged from 110–130 beats/ min at 37°C. On the third day, the number of myocytes increased to 3.0±0.1×105 cells/dish, and the myocyte beating rate increased to 130–150 beats/min. Such cultures were stable for up to 10 days. Myocytes were used on the third day of culture in the current experiments. The cells continued to beat spontaneously after loading with Fluo-3. During the ischemic period, the myocytes immediately stopped contracting after addition of hyperkalemic CP.
Effects of CP Ca2+ and Mg2+ concentrations on [Ca2+]i Figure 1 shows the time course for changes in [Ca2+]i, expressed as changes in fluorescence intensity of Ca2+-bound Fluo-3, during the ischemic and reoxygenation periods. In these studies, the Ca2+ concentration ([Ca2+]o) of the CP was varied, but Mg2+ was not included in the CP. The [Ca2+]i did not change during the first 20 min of ischemia, regardless of [Ca2+]o, but increased to 274±28% (n=46) of the control value in the 0.1 m [Ca2+]o CP group, 155±4% (n=34) in the 0.6 m [Ca2+]o CP group, 577±36% (n=48) in the 1.2 m [Ca2+]o CP group, and 209±23% (n=45) in the 2.4 m [Ca2+]o CP group after 60 min of ischemia. The
2+
[Ca ]i (% of control)
The data from at least 24 myocytes were used to determine [Ca2+]i. For the measurement of the recovery of the myocyte beating rate and the leakage of CPK and LDH, data obtained from six dishes of cultured myocytes were used. All values are expressed as the mean±standard error of the mean (±...). Significant effects were determined using the Kruskal–Wallis non-parametric analysis of variance. If significant differences were found, the multiple comparison procedure was used to identify which groups were significantly different. Moreover, the Pearson’s correlation coefficient was used to analyse correlations between [Ca2+]i, the beating rate recovery, and the leakage of CPK and LDH. Differences were considered significant if P<0.05.
Simulated ischemia (NaCN, glucose-free)
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Figure 1 Effects of the Ca2+ concentration in Mg2+-free CP on intracellular Ca2+ concentration ([Ca2+]i). Data shown represent the mean±... from 34 to 48 myocytes per group. (Χ), 0.1 m; (Ε), 0.6 m; (Η), 1.2 m; (Β), 2.4 m. (∗ P<0.01, comparing [Ca2+]i after 60 min of ischemia for 1.2 m group to [Ca2+]i for the other groups; † P<0.01, comparing the maximum [Ca2+]i during reoxygenation for 0.1 m group to [Ca2+]i for the other groups).
[Ca2+]i in the 1.2 m [Ca2+]o CP group was significantly greater than the other CP groups following 60 min (P<0.01). The [Ca2+]i initially increased in all groups during the subsequent reoxygenation period. The maximum [Ca2+]i during reoxygenation were 771±147, 166±5, 622±38, and 250±30% in the 0.1, 0.6, 1.2, and 2.4 m [Ca2+]o CP groups, respectively. The [Ca2+]i during reoxygenation in the 0.1 m [Ca2+]o group significantly increased to a greater extent compared to the other groups (P<0.01). For each [Ca2+]o, the [Ca2+]i after 60 min of ischemia and the maximum [Ca2+]i during reoxygenation varied with the Mg2+ concentration ([Mg2+]o) in the CP (Fig. 2). In 0.1 m [Ca2+]o CP, [Ca2+]i after 60 min of ischemia was significantly lower for CP containing 1.2 m Mg2+, 8 m Mg2+, and 16 m Mg2+ than CP free of Mg2+ (P<0.01). After perfusion with 0.1 m [Ca2+]o CP, the maximum [Ca2+]i during reoxygenation was also significantly lower for CP containing 1.2 m Mg2+ (115±18%), 8 m Mg2+ (166±13%), and 16 m Mg2+ (184±17%) than CP free of Mg2+ (P<0.01). However, the maximum [Ca2+]i during reoxygenation increased to a greater extent if the [Mg2+]o was greater than 1.2 m in the CP. In the 0.6 m [Ca2+]o CP group, [Ca2+]i after 60 min of ischemia at a [Mg2+]o of 1.2, 8, and 16 m were 134±14, 97±4, and 101±2%, respectively. [Ca2+]i after 60 min of ischemia at 8 m and 16 m [Mg2+]o were significantly lower than in cells perfused with CP without Mg2+ (P<0.01). The maximum [Ca2+]i during reoxygenation at a
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Calcium and Magnesium in Cardioplegia for Immature Hearts 200
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[Mg ]o (mM)
Figure 2 Relationship between intracellular Ca2+ concentration ([Ca2+]i) and Mg2+ concentration ([Mg2+]o) for each Ca2+ concentration ([Ca2+]o) CP. Closed histograms represent [Ca2+]i after 60 min of ischemia. The open histograms represent the maximum [Ca2+]i during reoxygenation. Data shown represent the mean±... from 24 to 48 myocytes per group. (∗ P<0.01, compared to [Ca2+]i after 60 min of ischemia when no Mg2+ was present; † P<0.01, compared to the maximum [Ca2+]i during reoxygenation when no Mg2+ was present; ‡ P<0.05 and § P<0.01 compared to the maximum [Ca2+]i during reoxygenation for 1.2 m [Mg2+]o).
[Mg2+]o of 1.2 m, 8 m, and 16 m were 148±16, 101±5, and 107±2%, respectively. The maximum [Ca2+]i during reoxygenation in the presence of 8 or 16 m Mg2+ was also significantly lower than if Mg2+ was absent (P<0.01). In the 1.2 m [Ca2+]o CP group, [Ca2+]i after 60 min of ischemia at 1.2 m [Mg2+]o, 8 m [Mg2+]o, and 16 m [Mg2+]o were significantly less than values obtained for CP without Mg2+ (P<0.01). The maximum [Ca2+]i during reoxygenation at 1.2 m [Mg2+]o, 8 m [Mg2+]o, and 16 m [Mg2+]o were also significantly lower than values for CP without Mg2+ (P<0.01). In the 2.4 m [Ca2+]o CP group, [Ca2+]i after 60 min of ischemia at a [Mg2+]o of 1.2, 8, and 16 m were 161±12, 144±24, and 118±17%, respectively. [Ca2+]i after 60 min of ischemia at 16 m [Mg2+]o was significantly lower than if CP contained no Mg2+ (P<0.01). The maximum [Ca2+]i during reoxygenation at a [Mg2+]o of 1.2, 8, and 16 m were 166±14, 155±26, and 104±12%, respectively. The maximum [Ca2+]i reoxygenation at 16 m [Mg2+]o CP was significantly less than if CP contained no Mg2+ (P<0.01).
Cardiac myocyte beating recovery after ischemia In the 0.1 m [Ca2+]o CP group, the beating rate recovery at 1.2, 8, and 16 m [Mg2+]o were better than CP without Mg2+ (P<0.01). In this group, there was no recovery of beating because hypercontracture had developed early during reoxygenation if there was no Mg2+ in the CP. In the 1.2 m [Ca2+]o CP group, the beating rate recovery at 8 and 16 m [Mg2+]o were significantly better than CP without Mg2+ (P<0.05). In the 0.6 and 2.4 m [Ca2+]o CP groups, in which the increased [Ca2+]i during ischemia and reoxygenation in Mg2+-free CP were minute, the beating rate recovery was not significant irrespective of the presence of extracellular Mg2+ (Table 1). In addition, with the exception of 1.2 m [Ca2+]o CP groups, beating rate recovery indicated a [Ca2+]o dependent increase at 0 m [Mg2+]o. There was a significant correlation between the maximum [Ca2+]i through the ischemia and reoxygenation periods and the beating rate recovery for all of the groups (r=−0.74, P<0.001).
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T. Ichiba et al. Table 1 Dose responses to Ca2+ and Mg2+ concentrations in CP Ca2+ and Mg2+ concentrations (m) Ca2+ 0.1 Mg2+0 1.2 8 16 P value Ca2+ 0.6 Mg2+0 1.2 8 16 P value Ca2+ 1.2 Mg2+0 1.2 8 16 P value Ca2+ 2.4 Mg2+0 1.2 8 16 P value
Beating rate recovery (%)
CPK leakage (mIU/dish)
LDH leakage (mIU/dish)
0 61±7∗ 54±5∗ 47±13∗ <0.01
23.6±1.0 15.0±0.3∗ 15.6±0.2∗ 17.0±0.6∗ <0.01
27.9±1.2 7.9±0.4∗ 8.6±0.4∗ 9.3±1.0∗ <0.01
80±2 89±8 90±6 85±18 ..
16.5±0.3 14.4±0.7 13.4±0.7 14.6±0.8 ..
11.8±0.2 9.2±0.6∗ 8.1±0.4∗ 8.8±0.5∗ <0.01
55±6 71±7 75±4† 80±15† <0.05
20.1±1.4 17.4±0.5 16.4±1.0† 15.7±0.6† <0.05
12.7±0.1 9.1±0.9∗ 8.8±0.4∗ 9.1±0.3∗ <0.01
97±10 98±2 95±3 97±17 ..
16.0±0.1 14.9±0.1 15.1±0.6 14.9±0.3 ..
8.8±0.2 8.8±0.6 7.9±0.4 8.2±1.1 ..
The beating rate recovery after 30 min of reoxygenation is expressed as a percentage of the beating rate before ischemia. CPK and LDH leakages represent the total quantity during both ischemia and reoxygenation. Values are expressed as mean±..., with n=6 dishes in each group. ∗ P<0.01, † P<0.05, compared to 0 m Mg2+ CP group for each Ca2+ concentration. ..=not significant (P[0.05).
Enzyme leakage from myocytes during ischemia and reoxygenation Table 1 shows the total amount of CPK and LDH released from myocytes during both the ischemia and reoxygenation periods for each group. In the 0.1 m [Ca2+]o CP group, the CPK and LDH leakages at a [Mg2+]o of 1.2, 8, and 16 m were significantly lower than at 0 m [Mg2+]o. In the 0.6 m [Ca2+]o CP group, the release of LDH at a [Mg2+]o of 1.2, 8, and 16 m were significantly lower than at 0 m [Mg2+]o. However, the release of CPK did not differ significantly in this group. In the 1.2 m [Ca2+]o CP group, CPK leakage at a [Mg2+]o of 8 and 16 m and LDH release at 1.2, 8 and 16 m [Mg2+]o were significantly lower than at 0 m [Mg2+]o. In the 2.4 m [Ca2+]o CP group, neither CPK nor LDH leakage were affected by the [Mg2+]o. The enzyme leakages were also significantly lower in the 0.6 and 2.4 m [Ca2+]o CP groups than in the 0.1 and 1.2 m [Ca2+]o CP groups when the CP contained no Mg2+ (not shown in Table 1). Ultimately, the leakage of CPK and
LDH was significantly less in groups in which the increase in [Ca2+]i during both ischemia and reoxygenation were prevented (the correlation between the maximum [Ca2+]i through the ischemia and reoxygenation periods and CPK: r=0.945, P<0.0001; between the maximum [Ca2+]i and LDH: r=0.862, P<0.0001).
The correlation between beating rate recovery and enzyme leakage There was a significant correlation noted between the beating rate recovery and the leakage of CPK (r=−0.835, P<0.0001) and LDH (r=−0.794, P<0.0001). In comparison in each [Ca2+]o CP group response to [Mg2+]o, the beating rate recovery correlated significantly with the leakage of CPK in the 0.1 and 1.2 m [Ca2+]o CP groups and the leakage of LDH in 0.1 m [Ca2+]o CP group. On the other hand, in comparison in each [Mg2+]o, there was a significant correlation between the beating rate
Calcium and Magnesium in Cardioplegia for Immature Hearts
recovery and the enzyme leakage (both CPK and LDH) only in 0 m [Mg2+]o.
Discussion Our investigation demonstrates that the [Ca2+]o in hyperkalemic CP influences [Ca2+]i and myocardial protection in immature cardiac myocytes. The effect of extracellular Ca2+ was significant, especially when there was no Mg2+ present in the CP. Further, an optimized [Mg2+]o in CP inhibited increases in [Ca2+]i and attenuated cell damage during ischemia and reoxygenation. However, a high [Mg2+]o relative to a low [Ca2+]o in CP, such as in the 0.1 m [Ca2+]o CP group, resulted in Ca2+ overload during reoxygenation. Therefore, a high [Mg2+]o may be detrimental if [Ca2+]o in CP is very low. In contrast, the effects of Mg2+ on myocardial protection may be minimal, if [Ca2+]o in CP is as high as 2.4 m.
Characteristics of Ca2+ handling in immature myocytes Calcium overload, resulting from the failure of intracellular Ca2+ homeostasis during ischemia and reperfusion, is one of the main causes of ischemic myocardial injury. Several potential mechanisms may be responsible for alterations in [Ca2+]i: (1) changes in Na+/Ca2+ exchange; (2) alterations in sarcolemmal voltage-gated Ca2+ channels; and (3) changes in the Ca2+ release channel in the sarcoplasmic reticulum (SR). Recent studies have demonstrated that immature myocytes have a poorly developed SR, and as a result are more dependent on Ca2+ influx across the sarcolemma to generate tension (Anderson, 1989; Chin et al., 1990; Kaufman et al., 1990). Moreover, Huynh et al. (1992) and Wetzel et al. (1993) have shown that voltagegated Ca2+ channels are relatively deficient in immature myocytes. To compensate, Na+/Ca2+ exchange increases in immature myocytes (Artman, 1992). Accordingly, it is believed that in immature myocytes, sarcolemmal Ca2+ influx pathways, especially the Na+/Ca2+ exchanger, are important in the control of [Ca2+]i during ischemia and reperfusion.
The effect of Ca2+ in CP on [Ca2+]i during ischemia and reoxygenation The results of this study show that alterations in the Ca2+ concentration of CP without Mg2+ result in various patterns of intracellular Ca2+ overload during ischemia and reoxygenation in immature
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myocytes. This suggests the existence of several mechanisms that may be responsible for Ca2+ overload. Na+/Ca2+ exchanger activity, which is important for [Ca2+]i handling in the ischemic immature myocyte, is dependent on the concentration gradients of both Na+ and Ca2+ across the sarcolemma. In addition, the exchanger is dependent on membrane potential because of the electrogenicity of the exchange (3:1 Na+/Ca2+ exchange). This is based on studies that have shown that Ca2+ influx via reversal of the exchanger is enhanced in depolarized membranes (Wetzel et al., 1995). As anaerobic metabolism increases in the myocyte during ischemia, protons (H+) accumulate in the cytoplasm. If the extracellular pH does not become acidic because of perfusion with CP, the activity of the Na+/ H+ exchanger is not inhibited during ischemia. As a result, H+ accumulating in myocytes during ischemia are exchanged with extracellular Na+. The Na+ which enters the cell via the Na+/H+ exchanger is subsequently exchanged with extracellular Ca2+ via the Na+/Ca2+ exchanger. In addition, reversal of the Na+/Ca2+ exchanger results in faster exchange because the membrane potential becomes relatively more depolarized by hyperkalemic CP during ischemia. Taken together, these changes result in intracellular Ca2+ overload. The glycocalyx on the sarcolemma plays an important role in determining the [Ca2+]o and membrane potential by binding extracellular Ca2+. The glycocalyx can therefore regulate the activity of the Na+/Ca2+ exchanger (Langer, 1984). As a result, [Ca2+]i during ischemia and reoxygenation may be regulated by the degree of binding of Ca2+ to the glycocalyx. In our experiments, the increases in [Ca2+]i during ischemia and reoxygenation were suppressed to a greater extent in myocytes perfused with 0.6 or 2.4 m [Ca2+]o CP compared to myocytes perfused with 0.1 or 1.2 m [Ca2+]o CP when Mg2+ was not present. We suggest that the following mechanisms are responsible for this effect. First, in 0.6 m [Ca2+]o without Mg2+ CP, the amount of Ca2+ binding to the glycocalyx does not reverse the Na+/ Ca2+ exchanger either during ischemia or during reoxygenation with Tyrode solution containing 1 m [Ca2+]o and 1 m [Mg2+]o. In 1.2 m [Ca2+]o CP, severe overload occurs during ischemia because the increased binding of Ca2+ to the glycocalyx reverses Na+/Ca2+ exchange during ischemia. Finally, in 2.4 m [Ca2+]o CP, although the amount of Ca2+ bound to the glycocalyx increases, the membrane potential is greater than in 1.2 m [Ca2+]o because of the presence of excess Ca2+. As
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a result, the degree of depolarization caused by hyperkalemic CP decreases during the ischemic period. This effect may decrease the reversed activity of the Na+/Ca2+ exchanger, resulting in attenuated increases in [Ca2+]i. In 0.1 m [Ca2+]o CP, severe Ca2+ overload occurred during reoxygenation, suggesting the existence of a mechanism different from those described above. When myocytes were perfused with a low [Ca2+]o CP, such as 0.1 m, the glycocalyx-sarcolemma complex may have been partially disrupted. This disruption of a Ca2+ “trap” significantly changes the free [Ca2+]o at the sarcolemma. Accordingly, myocytes perfused with 0.1 m [Ca2+]o CP experience increased Ca2+ influx during subsequent reperfusion with 1 m [Ca2+]o, because the reversal of Na+/Ca2+ exchanger activity is augmented by the rapid increase in [Ca2+]o at the sarcolemma during reperfusion (i.e. “calcium paradox”) (Zimmerman and Hulsmann, 1966). The [Ca2+]o of CP therefore affects several important factors: (1) the stability of the glycocalyx-sarcolemmal complex; (2) the amount of Ca2+ binding to the glycocalyx, which in turn determines the quantity of Ca2+ that can enter cells; and (3) the membrane potential which determines both the polarity and the activity of the Na+/Ca2+ exchanger.
The effect of Mg2+ on [Ca2+]i during ischemia and reoxygenation In the adult heart, CP that contain Mg2+ produce better post-ischemic ventricular function than those that do not contain Mg2+ (Hearse et al., 1978; Reynolds et al., 1989). The exact protective mechanism of Mg2+ is not known at this time, although several mechanisms have been suggested. The first is that Mg2+ acts as a cofactor for energy-requiring intracellular reactions. Specifically, Mg2+ regulates both cyclic AMP-dependent voltage-gated Ca2+ channels and Ca2+ release from the SR (Agus and Morad, 1991). Furthermore, intracellular Mg2+ is important for Na+/K+ ATPase activity, which promotes the active transport of Na+ out of the cell. The resulting decrease in intracellular Na+ suppresses Ca2+ influx by reversing the Na+/Ca2+ exchanger. However, the cell membrane has a relatively low permeability to Mg2+ (Flatman, 1984), and the concentration of intracellular Mg2+ is unlikely to decrease significantly during ischemia or hypoxia (Fry, 1986; Murphy et al., 1989). A second mechanism involves the ability of Mg2+ to act as a physiologic inhibitor of Ca2+
transport across the sarcolemma (Iseri and French, 1984). In our study, the addition of Mg2+ to CP significantly suppressed intracellular Ca2+ overload as well as the beating rate recovery and/or enzyme leakage during ischemia and reoxygenation, with the exception of the 2.4 m [Ca2+]o CP group. However, the addition of more than 8 m Mg2+ to 0.1 m [Ca2+]o CP resulted in an increase in [Ca2+]i during reoxygenation, and poorer myocardial protection. The protective effects of Mg2+ cannot be ascribed solely to Ca2+ channel inhibition, especially in immature myocytes, in which Ca2+ channels are relatively deficient. Langer (1984) has suggested that Mg2+ competes with Ca2+ for binding sites within the sarcolemmal–glycocalyx complex of the myocyte. As a result, the addition of Mg2+ to CP decreases the magnitude of the Ca2+ influx via Ca2+ channels or the Na+/Ca2+ exchanger during ischemia because of a decrease in the relative [Ca2+]o. Moreover, the Mg2+ in CP prevents severe Ca2+ overload during reoxygenation after perfusion with low [Ca2+]o CP (i.e. “calcium paradox”) by preserving the sarcolemmal–glycocalyx complex. In contrast, a high [Mg2+]o relative to a low [Ca2+]o increases the Mg2+ binding of the glycocalyx, and inhibits Ca2+ influx into the cell during ischemia. However, Mg2+ is the least effective of the divalent cations in displacing Ca2+ from binding sites (Langer, 1984). Therefore, during reoxygenation, the Mg2+ bound to the glycocalyx is readily displaced by Ca2+. This would increase the [Ca2+]o rapidly and increase the exchange of intracellular Na+ for Ca2+ during reoxygenation. In 0.6 m [Ca2+]o CP, a [Mg2+]o greater than 8 m did not suppress the [Ca2+]i increase. It may be that the addition of Mg2+ at concentrations greater than 16 m to CP causes increases in [Ca2+]i during reoxygenation. Accordingly, we hypothesize that [Mg2+]o affects the [Ca2+]i less in a high [Ca2+]o CP group such as 2.4 m than the other [Ca2+]o CP groups.
The relationship between [Ca2+]o and cardioprotection In our study, the recovery of the beating rate and the release of CPK and LDH were used as parameters of the cardioprotective effect of CP. The recovery of the beating rate improved, and the release of both CPK and LDH were less in groups in which the increases in [Ca2+]i were suppressed during ischemia and reoxygenation. We found a statistically significant correlation between [Ca2+]i and either
Calcium and Magnesium in Cardioplegia for Immature Hearts
beating rate recovery or enzyme release. This suggests that there is a close correlation between the cardioprotective effects of CP and the regulation of [Ca2+]i during ischemia and reoxygenation. When CP contained no Mg2+, especially in the 0.1 m [Ca2+]o CP group, the damage would be due to rapid, severe intracellular Ca2+ overload resulting in cell destruction by hypercontracture during reoxygenation. When [Mg2+]o was varied in each [Ca2+]o CP group, the recovery of beating rate improved and the release of CPK and LDH were less in groups in which the increases in [Ca2+]i were suppressed during ischemia and reoxygenation. The beating rate recovery and enzyme leakage were not dependent on [Mg2+]o in the 2.4 m [Ca2+]o CP group. We hypothesize the reason that in the 2.4 m [Ca2+]o CP group the suppressing effect of Mg2+ on the [Ca2+]i increase may have been less than in the other [Ca2+]o CP groups. On the other hand, in comparison in each [Mg2+]o, there was no significant correlation between the beating rate recovery and the enzyme leakage, with exception of 0 m [Mg2+]o. The reason is considered to be that for 1.2, 8 and 16 m [Mg2+]o CP groups, the [Ca2+]i varied with [Ca2+]o in CP less compared with 0 m [Mg2+]o CP group. Consequently, in those groups, the beating rate recovery and the enzyme leakage did not vary so significantly as this group statistically.
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similar to that in ischemia. Tyrode solution containing 1 m Ca2+ and 1 m Mg2+ was perfused during the reoxygenation period in all experiments, in spite of using the CP which have various concentrations of Ca2+ and Mg2+ during ischemia, because we were attempting to simulate the clinical situation; namely, reperfusion by blood containing about 1 m Ca2+ and 1 m Mg2+ after cardiac arrest.
Conclusion The current study demonstrates that both Ca2+ and Mg2+ in CP influence cardioprotective effects by regulating the intracellular Ca2+ concentration during ischemia and reoxygenation in immature myocytes. Mg2+ in CP suppressed increases in the [Ca2+]i during ischemia and subsequent reoxygenation, thereby conferring protection. However, too great a [Mg2+]o relative to low [Ca2+]o may cause cell injury through Ca2+ overload during reoxygenation, while the protective effects of [Mg2+]o are weak if CP contains high [Ca2+]o. These results indicate that these cations in CP determine the amount of Ca2+ that enters the cell via the sarcolemma. Further, this influx is determined by both Na+/Ca2+ exchange and the glycocalyx in immature myocytes. This study helps to elucidate the cardioplegic effect of Ca2+ and Mg2+ in CP for immature hearts.
Methodologic considerations
References In the present study, we used neonatal myocyte monolayers on the third day of culture as a model of immature myocardium. A well-defined myocyte culture system is useful in evaluating the direct effects of various stimuli, including ischemia or post-ischemic reperfusion (Orita et al., 1994), because the myocytes are not affected by other cell components such as endothelial cells or fibroblasts. Moreover, a monolayer of cells maintains a constant synchronous beating rate. We simulated ischemia using a glucose-free perfusion with sodium cyanide. Depletion of intracellular ATP was achieved by metabolic inhibition with the perfusion. Although intracellular ATP was not measured in the present study, other investigators have reported that ATP is reduced by approximately 50% in neonatal cardiomyocytes after 90 min of either sodium cyanide induced metabolic stress or true hypoxia (Iwaki et al., 1993). The previous data indicate that the phenomenon which occurs in the present study is
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