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Circulation. 1995;92:405-412

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(Circulation. 1995;92:405-412.)
© 1995 American Heart Association, Inc.

Articles

Magnesium Cardioplegia Enhances mRNA Levels and the Maximal Velocity of Cytochrome Oxidase I in the Senescent Myocardium During Global Ischemia

Presented at the 67th Scientific Sessions of the American Heart Association, Nov 14-17, 1994, Dallas, Tex.

Elizabeth A. Faulk, MD; James D. McCully, PhD; Narelle C. Hadlow, MD; Takuro Tsukube, MD; Irvin B. Krukenkamp, MD; Micheline Federman, PhD; Sidney Levitsky, MD

From the Division of Cardiothoracic Surgery and the Department of Pathology, New England Deaconess Hospital and Harvard Medical School, Boston, Mass.

Correspondence to Sidney Levitsky, MD, Division of Cardiothoracic Surgery, New England Deaconess Hospital, 110 Francis St, Suite 2C, Boston, MA 02215.


*    Abstract
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 *Abstract
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 down arrowMethods
down arrowResults
down arrowDiscussion
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Background The aged myocardium accumulates significantly more cytosolic calcium [Ca2+]i during ischemia, and functional recovery is more severely compromised as compared with the mature heart. Cardioplegia ameliorates these phenomena. The mechanism by which increased calcium accumulation reduces functional recovery in the senescent myocardium is unknown, but it has been suggested that futile calcium cycling in the mitochondria leading to depletion of ATP stores during normothermic global ischemia may be involved.

Methods and Results To investigate the effect of cardioplegia on mitochondrial calcium ([Ca2+]mt) accumulation and the expression of cytochrome oxidase I (COX I) during global ischemia, mitochondria were isolated from mature (age, 15 to 20 weeks) and aged (age >130 weeks) rabbit hearts after Langendorff perfusion. Five perfused heart groups were investigated: 30 minutes of global ischemia without treatment (control), with potassium (K, 20 mmol/L), magnesium (Mg, 20 mmol/L), or potassium and magnesium (K/Mg) cardioplegia. No significant difference in [Ca2+]mt was evident in mature hearts with any protocol. In aged hearts, [Ca2+]mt was increased in global ischemia but was ameliorated with Mg and K/Mg cardioplegia. COX I mRNA levels in aged hearts were lower in both control and global ischemia but were increased with cardioplegia. Maximal velocities for COX I were significantly increased with Mg cardioplegia both in the mature and the aged myocardium.

Conclusions K and/or Mg cardioplegia ameliorates [Ca2+]mt accumulation in aged hearts during normothermic global ischemia and increases COX I mRNA levels to a level not significantly different from that found in mature hearts.


Key Words: cardioplegia • calcium • aging


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMethods
down arrowResults
down arrowDiscussion
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We have shown previously that in the isolated perfused rabbit heart, the induction of warm global ischemia results in the rapid accumulation of ([Ca2+]i) and precedes the onset of ischemic contracture.1 2 The aged (<135 weeks) heart was shown to accumulate 30% more [Ca2+]i during 30 minutes of normothermic global ischemia than the mature heart (age, 15 to 20 weeks).3 4 5 We also have shown that increased [Ca2+]i during ischemia was correlated with reduced functional recovery of the myocardium and that these functional decrements were amplified in the aged heart as compared with the mature heart.3 4 5 The mechanism(s) contributing to this age-related injury have yet to be elucidated; however, the regulation of cellular calcium transport and high-energy phosphate preservation have been postulated to play a central role in determination of myocardial ischemic injury.6 7 8 9

Myocardial tissue is primarily aerobic, and its metabolism is closely dependent on oxygen, as confirmed by the abundance of mitochondria (30% of the total volume). The high energy requirement of the myocardium is almost exclusively met by mitochondrial oxidative phosphorylation.10 This leads to a high sensitivity of the myocardial cell to oxygen deficiency, and mitochondrial function is likely to play a central role in the molecular events leading to tissue damage occurring under the condition of ischemia.11 One mechanism that may contribute to the reduction of ischemic tolerance in the senescent myocardium may relate to the observed alterations in calcium homeostasis in the aging mitochondria.10 12 13 Previous studies in adult rat heart have indicated that an increase in [Ca2+]i precedes lethal myocardial injury and that this increase is associated with the depletion of cellular ATP.7 Investigation of myocardial energy metabolism with aging in intact hearts and isolated mitochondria have shown that maximal myocardial substrate oxidation rates decline approximately 20% from the mature heart to the senescent heart in the rat.14 In addition, studies in isolated mitochondria have shown that aging is associated with reduced oxidation rates.15 16 These data suggest that during ischemia and reperfusion, myocardial high-energy phosphate preservation may be compromised in the aged as compared with the mature heart.

The mechanism of action of increased calcium accumulation in reducing functional recovery in the senescent myocardium is unknown at present, but it is reasonable to suggest that futile calcium cycling in the mitochondria leading to depletion of ATP stores during normothermic global ischemia would have a deleterious effect on the myocyte.10 17 18 19 Recently we have shown that the use of magnesium-supplemented potassium cardioplegia preserves high-energy phosphates best in the aged heart. The mechanism of action of this cardioplegic protection remains to be elucidated.20

One enzyme that may be an indicator of impaired mitochondrial function is COX. COX is the terminal enzyme complex of the inner mitochondrial electron transport chain and has been shown to be vital in the production of high-energy phosphate.10 The activity of cytochrome oxidase has been shown to sharply decline in the latter part of life and may compromise high-energy phosphate preservation in the aged.21

We have shown previously that magnesium-supplemented potassium cardioplegia modulates [Ca2+]i in aged hearts during 30-minute normothermic global ischemia and enhances myocardial functional recovery.2 4 5 The mechanism of action of magnesium-supplemented potassium cardioplegia may involve modulation of mitochondrial functiona through the amelioration of [Ca2+]mt accumulation. In this report, we show that magnesium-supplemented potassium cardioplegia ameliorates [Ca2+]mt accumulation during normothermic global ischemia in the senescent myocardium and enhances COX I, Vmax, and mRNA levels. With the increased incidence of elderly patients as candidates for complex cardiac surgery, these findings may have important implications for reducing morbidity and mortality during cardiac surgery.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
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New Zealand White rabbits aged 15 to 20 weeks (mature, n=60) and >130 weeks (aged, n=60) were obtained from Millbrook Farm, Amherst, Mass. All animals were housed individually and provided with laboratory chow and water ad libitum. All experiments were approved by the New England Deaconess Hospital Animal Care and Use Committee and conformed to the US National Institutes of Health guidelines regulating the care and use of laboratory animals. All chemicals used were of electrophoresis grade or ultrapure quality.

Surgical Preparation and Perfusion
All rabbits were anesthetized with sodium pentobarbital (Nembutal, 100 mg/kg IV) and heparin (200 U/kg IV). The heart was excised and placed in a 4°C bath of Krebs-Ringer solution containing (mmol/L) NaCl 100, KCl 4.7, CaCl2 1.7, MgSO4 1.2, NaHCO3 25, KH2PO4 1.1, glucose 11.5, sodium pyruvate 4.9, and sodium fumarate 5.4 equilibrated with 95% O2 and 5% CO2 (pH 7.4 at 37°C), in which spontaneous beating ceased within a few seconds. Polyethylene cannulas were advanced into the main pulmonary artery, the right superior vena cava, and the left atrium via the pulmonary vein, respectively, and held in place by sutures. The inferior and left superior vena cavae were closed near their insertion into the right atrium, and the left atrium was opened. A latex balloon containing a catheter-tipped transducer (Millar Instruments, Inc) was inserted into the left ventricle and held in place by a purse-string suture. The volume of the water-filled balloon was maintained at a constant physiological end-diastolic pressure in a range of 5 to 10 mm Hg with use of a calibrated microsyringe. The aorta was cannulated with a metal cannula, and the heart was subjected to Langendorff retrograde perfusion at a constant pressure of 75 cm H2O at 37°C. Left ventricular pressure was recorded, and an ECG was obtained with electrodes placed on the epicardial surface of the right ventricle to monitor equilibrium. The heart was placed in the water-jacketed chamber, and myocardial temperature was maintained at 37°C. After 30 minutes of Langendorff retrograde circulation for equilibrium, the hearts were used in the following protocols.

Measurement of [Ca2+]i Accumulation
The fluorescent calcium indicator fura-2 was used to measure quantitatively the [Ca2+]i accumulation. After 30 minutes of Langendorff retrograde circulation for equilibrium, background fluorescence and hemodynamic data of left ventricular pressure were recorded as control data. The heart then was loaded with 2.5 µmol/L fura-2 in Krebs-Ringer solution and recirculated for 15 minutes. After the loading process, the heart was perfused for 30 minutes with Krebs-Ringer solution to wash out unincorporated fura-2 AM. All of the effluent from the myocardium, including the fluorescent dye solution, was drained through the cannulas in order to keep the surface of the heart dry during the experiment. Fura-2 epifluorescence (510 nm) from the epicardial surface of the left ventricle was measured using an in-house spectrofluorescence system that supplied rapidly alternating excitation wavelengths (340 nm, 380 nm) to the isolated perfused heart and allowed for the quantitative determination of [Ca2+]i from the ratio of emission induced by the two excitation wavelengths.4 5 22 The fura-2 fluorescence ratio was calculated as previously described.4 5 22

Cardioplegia
The effects of cardioplegia on [Ca2+]i during 30 minutes of normothermic global ischemia were investigated in four cardioplegic groups for both mature and aged hearts. After 30 minutes of equilibrium perfusion, normothermic global ischemia was achieved by clamping the ascending aorta for 30 minutes. Tissue temperature was maintained at 37°C throughout the experimental protocols. The four cardioplegic groups investigated were (1) hearts subjected to ischemia without cardioplegia (global), (2) hearts subjected to ischemia after potassium cardioplegia (K, 20 mmol/L KCl), (3) hearts subjected to ischemia after magnesium cardioplegia (Mg, 20 mmol/L MgSO4), and (4) hearts subjected to ischemia after a combination of potassium and magnesium cardioplegia (K/Mg, 20 mmol/L each KCl and MgSO4). Cardioplegic solutions were perfused at a constant pressure of 75 cm H2O at 37°C for 5 minutes before the onset of ischemia. Control hearts were perfused for 65 minutes with Krebs-Ringer solution at a constant pressure of 75 cm H2O at 37°C. The hearts were rapidly frozen in liquid nitrogen; experimental protocols were followed to allow for subsequent determinations.

Comparison of Wet and Dry Weights
Frozen samples from all experimental groups were weighed (wet weight) and dried at 80°C for 24 hours for reweighing (dry weight) and then were used for the determination of wet/dry weight ratios, using previously described methods.4 5

Isolation of Mitochondria
Mitochondria were isolated by differential centrifugation according to the method of Welter et al.23 All procedures were performed at 4°C unless otherwise stated. Mitochondria were isolated under conditions that favored retention of endogenous calcium and magnesium, without the use of albumin or EDTA.24

Determination of [Ca2+]mt
Mitochondrial pellets were suspended in 1% hydrochloric acid and sonicated with the use of an ultrasonic homogenizer (36260 series, Cole-Palmer). Quantitative colorimetric determinations were carried out with the use of o-cresolphthalein complexone.25

Isolation of mtDNA
mtDNA was isolated according to the method of Welter et al.23 mtDNA concentration was determined by method according to Burton.26

Determination of Mitochondrial Magnesium
Mitochondrial magnesium content was determined by sonication, followed by quantitative colorimetric assay with use of the calmagnite reaction.27

Determination of COX Vmax and Km
Cytochrome oxidase activity was determined by methods described by Sohal.28 The decrease in absorbance at 550 nm was monitored for 10 minutes. Values were recorded and calculated at 1-minute intervals by the Ultrospec II (LKB) fitted with the ENZYME KINETICS software package (Pharmacia).

Isolation of RNA: Northern Hybridization
Total cellular RNA was isolated by the method of Chomzynski and Sacchi29 and fractionated on a 1% agarose gel containing 3% formaldehyde, 0.02 mol/L MOPS, pH 7.4, (3-[N-morpholino] propanesulfonic acid, and 0.001 mol/L disodium EDTA.30 The RNA was visualized by ethidium bromide staining and then was transferred to nitrocellulose overnight with 10x SSC (pH 7.0). COX I mRNA levels were detected using a 1-kb, EcoRI insert from the human COX I clone (American Type Culture Collection, ATCC). ATP synthase mRNA levels were detected using a 1.6-kb, EcoRI insert from the human ATP synthase clone (ATCC). The 2.0-kb insert from the ß-actin "housekeeping gene" cDNA (Clontech Laboratories) was used as a control. Each probe, COX I, ATPsynthase, and ß-actin, was successively hybridized on the same nitrocellulose membrane to detect transcripts. This sequence was repeated on four separate membranes (n=4). Isolated and purified cDNA fragments were labeled with 32Pd-ATP according to the method described by Feinberg and Vogelstein.31 Free nucleotides were removed by Sephadex G-50 (Pharmacia) column chromatography.32

Semiquantitative Analysis
Semiquantitative analysis of autoradiographies was performed with the use of an LKB Ultrascan XL laser densitometer (Pharmacia LKB). The integral for each blot was calculated with use of the LKB GelScan XL software program for one-dimensional analysis.

Statistical Analysis
Statistical analysis was performed using the STATVIEW II software package. All results are presented as mean±SEM. Experimental differences were determined by a one-way ANOVA and the Scheffé test. Significance is claimed whenever the confidence level is greater than 95% (P<.05).


*    Results
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 up arrowIntroduction
 up arrowMethods
*Results
down arrowDiscussion
down arrowReferences
 
Effects of Cardioplegia on Dry Weight/Wet Weight Ratios After 30 Minutes of Normothermic Global Ischemia in the Mature and Aged Myocardium
The effects of cardioplegia on dry weight/wet weight ratios after 30 minutes of normothermic global ischemia in the mature and aged myocardium are shown in Table 1Down. No significant difference in dry weight/wet ratios was found between mature and aged hearts with any cardioplegic treatment groups when compared with controls.


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  		Table 1. Effects of Cardioplegia on Dry Weight/Wet Weight Ratios

mtDNA Concentration
To allow for standardization of results and to account for possible differences in mitochondrial populations in the mature and aged heart, mtDNA concentration per gram wet weight was measured.36 Our results (Table 2Down) indicate that mtDNA concentrations per gram wet weight were not statistically different between mature and aged hearts in control and that no significant change in mtDNA concentration occurred as the result of perfusion with or without cardioplegia. These results allowed for the use of mtDNA to provide a basis for the comparison of intraorganelle cation concentration.


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  		Table 2. Effects of Cardioplegia on Mitochondrial DNA Concentration (µg/g Tissue Wet Weight)

Effects of Cardioplegia on Mitochondrial Magnesium Accumulation After 30 Minutes of Normothermic Global Ischemia in the Mature and Aged Myocardium
To ensure that observed differences in [Ca2+]mt were not the result of differences in mitochondrial magnesium uptake, [Mg2+]mt in mitochondrial pellets was determined. Our results shown in Table 3Down indicate that [Mg2+]mt expressed as micromoles per liter per milligram of mtDNA was not significantly different between mature and aged hearts in control and were not altered by cardioplegic treatment when compared with controls.


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  		Table 3. Effects of Cardioplegia on Mitochondrial Magnesium (µmol/L per µg mtDNA)

Effects of Cardioplegia on [Ca2+]i and [Ca2+]mt Accumulation After 30 Minutes of Normothermic Global Ischemia in the Mature and Aged Myocardium
The effects of cardioplegia on [Ca2+]i and [Ca2+]mt during 30 minutes of normothermic global ischemia in the mature and aged myocardium are illustrated in Figs 1Down and 2Down. Ca2+]i was significantly increased (P<.05) from 178.7±26.5 to 393.6±28.0 nmol/L in mature hearts subjected to normothermic global ischemia without cardioplegia (global) as compared with preischemic (Cont, Fig 1Down). The use of K cardioplegia reduced [Ca2+]i accumulation to 300.9±10.6 nmol/L (P<.05), but Mg or K/Mg cardioplegia was found to completely inhibit [Ca2+]i accumulation (198.7±25.4 and 182.3±21.1 nmol/L, respectively; P<.05). No significant difference in [Ca2+]i accumulation was found between Mg and/or K/Mg cardioplegia.



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  		Figure 1. Bar graphs show effects of cardioplegia on cytosolic and mitochondrial calcium accumulation after 30-minute normothermic global ischemia in mature (20 weeks) rabbit hearts. Cytosolic calcium (nmol/L Ca2+) accumulation was measured using fura-2. Mitochondrial calcium (nmol/L Ca2+/µg mtDNA) accumulation was measured by spectrophotometric analysis and expressed as the percent increase above control, which was assigned as 100%. Cont indicates control hearts, perfused 65 minutes with Krebs-Ringer solution at 37°C at 75 mm Hg; Global, hearts subjected to 30-minute normothermic global ischemia without cardioplegia; K, hearts subjected to 30-minute normothermic global ischemia after potassium cardioplegia; Mg, hearts subjected to 30-minute normothermic global ischemia after magnesium cardioplegia; and K/Mg, hearts subjected to 30-minute normothermic global ischemia after magnesium-supplemented potassium cardioplegia. All results are shown as mean±SEM; n=6 for each group. *P<.05.



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  		Figure 2. Bar graphs show effects of cardioplegia on cytosolic and mitochondrial calcium accumulation after 30-minute normothermic global ischemia in aged (>135 weeks) rabbit hearts. Cytosolic calcium (nmol/L Ca2+) accumulation was measured using fura-2. Mitochondrial calcium (nmol/L Ca2+/µg mtDNA) accumulation was measured by spectrophotometric analysis and expressed as the percent increase above control, which was assigned as 100%. Cont indicates control hearts, perfused 65 minutes with Krebs-Ringer solution at 37°C at 75 mm Hg; Global, hearts subjected to 30-minute normothermic global ischemia without cardioplegia; K, hearts subjected to 30-minute normothermic global ischemia after potassium cardioplegia; Mg, hearts subjected to 30-minute normothermic global ischemia after magnesium cardioplegia; and K/Mg, hearts subjected to 30-minute normothermic global ischemia after magnesium-supplemented potassium cardioplegia. All results are shown as mean±SEM; n=6 for each group. *P<.05.

[Ca2+]mt accumulation expressed as the percent increase above control during 30 minutes of normothermic global ischemia in the mature myocardium also is illustrated in Fig 1Up. No significant difference in [Ca2+]mt accumulation was found to occur in the mature heart during 30 minutes of normothermic global ischemia with or without the use of cardioplegia. No difference in [Ca2+]i was found between mature (178.7±26.5 nmol/L) and aged (207.0±28.7 nmol/L) hearts during the preischemic perfusion period. In aged hearts (Fig 2Up) subjected to normothermic global ischemia without cardioplegia (global), [Ca2+]i accumulation was increased to 501.0±50.5 nmol/L (P<.05), a level approximately 30% above that observed in mature hearts (P<.05). The use of K cardioplegia reduced [Ca2+]i accumulation to approximately 75% of that obtained in hearts subjected to ischemia without cardioplegia in aged hearts (365.2±27.7 nmol/L, P<.05), whereas Mg or K/Mg cardioplegia was found to attenuate [Ca2+]i accumulation (261.3±26.7 and 262.3±25.2 nmol/L, respectively; P<.05). No significant difference in [Ca2+]i accumulation was found between Mg and/or K/Mg cardioplegia.

[Ca2+]mt accumulation during 30 minutes of normothermic global ischemia in the aged heart (Fig 2Up) was significantly increased above control in global ischemia (166%, P<.05) and with K cardioplegia (143%, P<.05). The use of Mg and K/Mg cardioplegia ameliorated these effects. No significant difference in [Ca2+]mt accumulation was found between Mg and/or K/Mg cardioplegia.

Effects of Cardioplegia on COX I mRNA Levels After 30 Minutes of Normothermic Global Ischemia in the Mature and Aged Myocardium
A representative Northern blot showing the effect of cardioplegia on COX I mRNA levels after 30 minutes of normothermic global ischemia in the mature and aged myocardium is shown in Fig 3Down. Analysis with the use of scanning laser densitometry (Fig 4Down) indicated that mRNA levels of COX I remained unchanged in all mature protocols (control, normothermic global ischemia, or normothermic global ischemia treated with high [20 mmol/L] K cardioplegia, high [20 mmol/L] Mg cardioplegia, or high K/Mg cardioplegia).



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  		Figure 3. Effects of cardioplegia on COX I mRNA levels after 30-minute normothermic global ischemia (GI) in mature and aged rabbit hearts (see Fig 2Up for other abbreviations). Top, Total ventricular RNA (10 µg) was loaded onto a 1% agarose-MOPS-formaldehyde gel and the RNA was visualized by ethidium bromide staining. Ribosomal 28S and 18S RNA markers are indicated. Middle, Northern blot analysis of the RNA gel shown in top panel. Hybridization was performed using a 1-kb, EcoRI insert from the human COX I clone. The Northern blot was rehybridized with ATP synthase mRNA levels (not shown) and the ß-actin "housekeeping gene" cDNA (shown in bottom panel). The use of cardioplegia was found to have no effect on COX I mRNA levels in the mature heart. In the aged heart, the use of cardioplegia was found to increase COX I mRNA levels significantly. No changes in ATP synthase or ß-actin mRNA levels were found between mature and aged hearts with or without cardioplegia.



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  		Figure 4. Bar graph depicts semiquantitative analysis with use of scanning laser densitometry of COX I mRNA levels in the mature and aged rabbit heart after 30-minute normothermic global ischemia. No difference in COX I mRNA levels were found with or without cardioplegia in mature hearts. In aged hearts, COX I mRNA levels were significantly decreased as compared with mature hearts in control and global. The use of cardioplegia significantly increased COX I mRNA levels in aged hearts to a level not significantly different from that of mature hearts. All values shown are mean±SEM; n=4 for each group. *P<.05; **P<.05 vs aged control and global.

In the aged myocardium, COX I mRNA levels were found to be significantly decreased (P<.05) in both control and global ischemia, with mRNA levels only approximately 50% that found in mature hearts (0.183±0.028 versus 0.364±0.029 arbitrary units in control for aged and mature, respectively) and after 30 minutes of normothermic global ischemia (0.178±0.019 versus 0.423±0.013 arbitrary units, P<.05 for aged and mature, respectively). The use of cardioplegia was found to increase COX I mRNA levels significantly such that there was no significant difference between mature and aged hearts. No significant difference between cardioplegic treatments was observed. COX I mRNA levels remained elevated in aged hearts after 30 minutes of normothermic reperfusion (results not shown).

No difference in ATP synthase mRNA levels was found between mature and aged hearts or with any cardioplegic treatment (results not shown). No difference in ß-actin mRNA levels was found between mature and aged hearts or with any cardioplegic treatment (Fig 3Up).

Effects of Cardioplegia on COX Vmax After 30 Minutes of Normothermic Global Ischemia in the Mature and Aged Myocardium
The effects of cardioplegia on COX Vmax after 30 minutes of normothermic global ischemia in the mature and aged myocardium are shown in Fig 5Down. No significant difference in COX Vmax (micromoles per liter of cytochrome c oxidized per minute) was found between mature and aged hearts in control. In mature hearts COX Vmax was significantly increased above global with Mg cardioplegia. COX Vmax in global ischemia, K cardioplegia, and K/Mg cardioplegia in mature hearts was not significantly different from control.



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  		Figure 5. Bar graph shows effects of cardioplegia on Vmax of COX activity after 30-minute normothermic global ischemia in the mature and aged rabbit heart. COX activity was measured as micromoles per liter of cytochrome c oxidized per minute with use of 20 µg mitochondrial protein in each reaction mixture. Magnesium cardioplegia was found to significantly increase COX I Vmax. All results are shown as mean±SEM; n=6 for each group. *P<.05 vs global; **P<.05 vs control.

In aged hearts after 30 minutes of normothermic global ischemia, COX Vmax was found to be significantly decreased (P<.05) but was significantly increased above global (P<.05) with Mg cardioplegia. K and K/Mg cardioplegia increased COX Vmax above that found in global ischemia and were not significantly different from control.


*    Discussion
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 up arrowMethods
up arrowResults
*Discussion
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Ischemia has been shown to increase [Ca2+]mt accumulation, resulting in decreased mitochondrial cytochrome and respiratory activity.17 Increased [Ca2+]mt accumulation destabilizes the inner mitochondrial membrane and causes the inner membrane pore to open, permitting further movement of cations across the mitochondrial membrane.10 The opening of these pores renders the mitochondrion incapable of making ATP and has been suggested as a key event in the process leading to myocardial cell death.33 In previous reports, we have shown that in the isolated perfused rabbit heart, the induction of warm global ischemia results in the rapid accumulation of [Ca2+]i and precedes the onset of ischemic contracture.1 34 In a series of experiments using newborn (age, 3 to 5 days) and adult (age, 4 to 5 months) rabbit hearts, an age-related susceptibility to ischemia/reperfusion injury was observed.3 35 When challenged with 30 minutes of warm global ischemia followed by 30 minutes of normothermic reperfusion, newborn hearts returned rapidly to preischemic cardiac function, and myocardial high-energy phosphate stores were restored. In the adult hearts subjected to the same protocol, functional recovery was significantly delayed, and the recovery of high-energy phosphate stores was much slower in returning to preischemic levels.36 These age-related differences in functional recovery were correlated with lower [Ca2+]i accumulation during ischemia in the newborn as compared with the adult myocardium.35

In this report, we show that 30 minutes of normothermic global ischemia results in the rapid accumulation of [Ca2+]i. In mature hearts, increased [Ca2+]i was not associated with increased [Ca2+]mt (Fig 1Up). In the aged heart, increased [Ca2+]i was associated with increased [Ca2+]mt (Fig 2Up). The use of Mg or Mg/K cardioplegia modulated [Ca2+]mt accumulation such that no significant difference in [Ca2+]mt was found between mature and aged hearts after 30 minutes of normothermic global ischemia (Figs 1Up and 2Up). These data would indicate that in the aged heart, [Ca2+]mt accumulation is altered to a greater degree than in the mature heart. The use of cardioplegia in the aged heart would appear to ameliorate the age-related differences in [Ca2+]mt accumulation.

Several lines of evidence indicate that mitochondrial efficiency decreases with age. In the kidney, ischemia and reperfusion have been shown to damage mitochondrial structure and impair respiratory function after 45 minutes of renal ischemia.17 The investigators reported that reperfusion was associated with altered mitochondrial mRNA expression and speculated that enhanced mitochondrial mRNA expression would aid in renal recovery from 45 minutes of ischemia.17 Nishimura et al37 have shown that in the isolated perfused rat heart, the effect of anoxia followed by reoxygenation in isolated mitochondria induced mitochondrial enzyme release and that the magnitude of the release was dependent on the duration of the anoxic period and the concentration of cytosolic ATP. Nohl et al19 have shown that in isolated heart mitochondria treated with ischemia and reperfusion, there was incomplete collapse of the transmembrane proton gradient and the impairment of respiration-linked ATP generation. Our results reported previously20 and those reported herein would agree with these findings and suggest that the use of Mg and/or Mg-supplemented cardioplegia may act to ameliorate these age-related phenomena associated with reduced functional recovery after surgically induced ischemia in the aged heart.

Previous investigators have reported that magnesium included in K cardioplegia (St Thomas' Hospital) was beneficial to coronary flow and aided in the reduction of myocardial enzymatic leakage in the ischemic and reperfused heart.38 Tosaki et al39 also have shown that by increasing extracellular magnesium, there is a reduction in reperfusion-induced ventricular fibrillation and ventricular tachycardia in rat hearts. Recently, Steenbergen et al7 have examined the relationship between ATP depletion, [Ca2+]i concentration, and lethal myocardial ischemic injury in the perfused rat heart with the use of nuclear magnetic resonance and reported that high magnesium (16 mmol/L) arrest delayed [Ca2+]i accumulation and ATP depletion longer than high potassium (30 mmol/L) arrest during normothermic ischemia in the mature rat heart.

High-energy phosphates are required for the proper maintenance of the heart as an aerobic organ. Under normal (nonischemic) conditions, calcium transport from the myocyte during diastole occurs against an electrochemical gradient requiring the use of ATP-dependent calcium transport mechanisms.6 36 The induction of normothermic global ischemia has been shown to rapidly reduce cellular high-energy phosphates. This reduction occurs in the ischemic myocardium through the continuance of a number of reactions that persist during ischemia and include the energy-dependent cellular transport mechanisms and enzymatic reactions.6 36 Previous investigation has shown that rabbit hearts subjected to normothermic global ischemia are rapidly depleted of ATP tissue stores and that the accumulation of [Ca2+]i is associated with or precedes the changes in cellular high-energy phosphates.40

In the myocardium, magnesium has been shown to act as a physiological calcium blocker as well as being required as an essential cofactor complexed to ATP and other adenine nucleotides for energy transfer reactions.41 42 It also has been shown that high magnesium concentrations in the extracellular space inhibit calcium entry into the cell by displacing calcium from its binding sites in the sarcolemmal membrane.43

The mechanism of action of Mg-supplemented cardioplegia remains to be described, but one possibility for this cardioprotection is the inhibitory action of magnesium. To date, ruthenium red and magnesium are the only two reported agents to block calcium influx via the mitochondrial uniporter.44 Benzi and Lerch45 have shown that postischemic perfusion with ruthenium red, a hexavalent dye that inhibits [Ca2+]mt uptake, significantly decreased oxygen consumption in the ischemic heart and enhanced contractile function.

It has been speculated that mitochondrial "futile calcium cycling," an energy-dependent process requiring ATP to transport calcium against the electrochemical gradient out of the mitochondrion, utilizes needed ATP required by the ischemic cell for more necessary functions and that inhibition of this process would be of benefit to myocardial functional recovery.24

The mechanism leading to increased [Ca2+]mt accumulation in the aged as compared with the mature myocardium is unknown; however, it may be speculated that alterations in mitochondrial oxidative capacity may account for at least part of the difference observed. Previous investigators have reported an age-related decline in mitochondrial energy production, possibly as a result of decreased repair or resynthesis of mitochondrial enzymes.46 Gadeleta et al have shown in the senescent rat heart (and brain) that while mtDNA copy number per cell remains stable, there is a reduced steady state level of mtDNA transcripts compared with the adult rat and that this decrease is due to reduced mitochondrial RNA synthesis in the aged rat heart. It is reasonable to speculate that increased [Ca2+]mt accumulation in the aged heart may be a consequence of the reduced synthesis of mitochondrial enzymes required for maintenance of [Ca2+]mt homeostasis.

Support for this hypothesis may be seen in the response of COX Vmax to global ischemia. Our results indicate that COX Vmax in mature and aged hearts is not significantly different at control (preischemia), but, after 30-minute global ischemia, COX Vmax in aged hearts is decreased significantly as compared with mature hearts (Fig 5Up). Our data also indicate that in the aged heart, COX I mRNA levels remain decreased as compared with the mature heart during global ischemia. These results would support the hypothesis that the mitochondria of the aged heart is compromised such that resynthesis of oxidative enzymes is attenuated. The increase in COX I mRNA levels and the associated increase in COX Vmax with the use of Mg cardioplegia would indicate that this age-related modulation in mitochondrial response may be reversible.

Aging in the myocardium would appear, from our data, to compromise the ability of the mitochondria to regulate calcium transport during normothermic global ischemia. The effects of aging and normothermic global ischemia on the aged myocardium would also appear to decrease mitochondrial COX I mRNA transcript levels, which possibly would contribute to the diminishment of high-energy phosphates. Our results show that the use of cardioplegia increased COX I mRNA levels in the aged heart to a level not significantly different from that found in the mature heart (Figs 3Up and 4Up). COX activity (Vmax) also was increased with cardioplegia above that found in global ischemia in the aged heart.

An association between increased [Ca2+]i and [Ca2+]mt accumulation in the aged myocardium appears to exist and is associated with reduced functional recovery in the aged as compared with the mature heart. The use of Mg-supplemented cardioplegia appears to ameliorate these factors and allows for enhanced functional recovery, possibly through improved mitochondrial function. These findings may have important implications in reducing morbidity and mortality during cardiac surgery in the aged heart.


*    Selected Abbreviations and Acronyms
 
[Ca2+]i = cytosolic calcium
[Ca2+]mt = mitochondrial calcium
COX = cytochrome oxidase
mtDNA = mitochondrial DNA<\/.>


*    Acknowledgments
 
This study was supported by the National Institutes of Health (HL-29077).


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
up arrowDiscussion
*References
 

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