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Cardiovascular Research 1997 34(3):547-556; doi:10.1016/S0008-6363(97)00058-8
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

Alteration in the control of mitochondrial respiration by outer mitochondrial membrane and creatine during heart preservation

Laurence Kay, Zoya Daneshrad, Valdur A. Saks1,1 and André Rossi*

Laboratoire de Bioénergétique, Université Joseph Fourier, BP 53-38041, Grenoble Cedex 9, France

* Corresponding author. Tel.: +33 04 76 51 46 70; fax: +33 04 76 51 42 18.

Received 4 October 1996; accepted 14 January 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: This study aimed to evaluate the nature and extent of mitochondrial alterations during heart preservation. Methods: Rat hearts, isolated after cardioplegia in situ, were preserved for 6 or 15 h at 4°C either by immersion in cardioplegic solution or by low-flow perfusion (0.3 ml/min) with cardioplegic solution. The energy state of hearts at the end of preservation was determined by 31P-NMR spectroscopy and functional recovery was evaluated on reperfusion. Variables of mitochondrial respiration (maximal rate of respiration in the presence of ADP={Delta}Vmax, apparent Km for ADP, effect of creatine) were evaluated on skinned fibers and compared with those determined in controls and in hearts subjected to 1 hour of ischemia at 37°C. Results: Serious mitochondrial alterations were detected in fibers from 15 h immersed hearts: decrease of {Delta}Vmax and of apparent Km for ADP, loss of the stimulatory effect of creatine, and disruption of the outer mitochondrial membrane. The extent of alterations was more accentuated in fibers from normothermic ischemic hearts, in which some damage of the inner mitochondrial membrane also occured. In fibers from hearts preserved for 6 h, no significant changes in mitochondrial variables could be detected. When the hearts were preserved under low-flow perfusion for 15 h, only the stimulatory effect of creatine on respiration was significantly decreased. The extent of the loss of the stimulatory effect of creatine paralleled the accumulation of inorganic phosphate (Pi) during preservation and the decrease in left ventricular function on reperfusion. Conclusions: Alterations related to the outer membrane and the intermembrane space are among the earliest signs of damage to mitochondrial function during heart preservation. These alterations could be attributed to the swelling of mitochondria under the effect of Pi. The determination of mitochondrial parameters in biopsy samples could allow a simple and rapid evaluation of energy-producing and transfer capacities of the myocardium.

KEYWORDS Heart preservation; Myocardial ischemia; Skinned fibers; Mitochondria, outer membrane; Creatine kinase; Rat, heart


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
For clinical heart transplantation, donor hearts are stored for several hours by immersion in a cold cardioplegic solution (4–10°C). Functional alterations are noticed when the duration of heart preservation exceeds 4–6 h. The reasons for the poor mechanical recovery of hearts after long-term hypothermic ischemia are not very clear. This decrease in mechanical function can result from alterations at various levels: contractile machinery, calcium metabolism, energy balance, sarcolemmal function etc. Our interest was directed to the alteration in energy balance of the heart during preservation. Since the main site of ATP production is mitochondria, it is of great interest to know the alteration in mitochondrial function after heart preservation. The impairment of mitochondrial function during ischemia at 37°C has already been well documented by numerous experimental studies [1–8]. The mitochondrial modifications which occur at low temperature (4°C) during long periods of time were less studied [9, 10]. Moreover, these studies, performed on isolated mitochondria, did not take into consideration the step of energy transfer by phosphocreatine and the creatine kinase system.

From a clinical point of view, evaluation of the capacity of the heart to resume satisfactory mechanical function at the end of the preservation period remains one of the main issues in heart transplantation. When considering the energetic status of the heart, the high-energy phosphate (HEP) content may be a useful indicator. Such variables can be evaluated quite easily on muscle biopsies, or using non-invasive NMR spectroscopy. However, it is known from experimental studies that there is not always a direct relationship between HEP content and recovery of cardiac function following ischemia [11, 12]. The energetic potentialities, the production and transfer of energy are probably more indicative of heart viability.

The present experiments were therefore focused on the changes that occur at the level of mitochondrial ATP production and transfer in the cardiomyocyte after hypothermic heart preservation. For this purpose, we used the permeabilized cardiac fiber technique for the determination of mitochondrial respiration rates. This technique has been developed by Veksler et al. [13] and Saks et al. [14, 15] for studies of cellular respiration and factors controlling oxidative phosphorylation. It has also been successfully used for experimental and clinical studies of skeletal and heart muscles [16–19]. A significant advantage of this method is that the whole cellular population of mitochondria is analysed in the cells from only 5–10 mg of tissue.

Mitochondrial respiration was evaluated for various modes and durations of heart preservation. The rat hearts were stored for 6 or 15 h by immersion in a cardioplegic solution, which is the current clinical process, or for 15 h by low-flow perfusion (0.3 ml/min), which improved cardiac recovery [20, 21]. Thus, the extent of mitochondrial alterations could be evaluated in fibers from hearts which resumed a variable contractile function on reperfusion, depending on the mode and duration of preservation.

The results show that the most sensitive indicators of ischemic damage and of the efficiency of heart protection and preservation procedures are: the respiration rate controlled by coupled creatine kinase activity and, to some extent, the functional state of the outer mitochondrial membrane.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Experimental protocols
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985).

Female Wistar rats weighting 200–250 g were anaesthetized with sodium pentobarbital (50 mg/kg body wt, i.p.) and treated with heparin (1500 IU/kg body wt, i.v.). The animals were divided into 6 groups.

Controls: The hearts were quickly removed and immersed in solution A (see below) at 4°C for fiber preparation (Cont.).

Ischemia: The hearts were quickly removed and immersed in physiological solution (in mM): NaCl 129, KCl 5.6, MgCl2 2.4, CaCl2 0.5, NaHCO3 21, glucose 11, at 4°C. Then they were subjected to global ischemia at 37°C for 1 h, kept in individual polyethylene bags immersed in water at 37°C (Isch.).

Heart preservation: The rats were intubated and ventilated with air before being thoracotomized. The cardiac arrest was induced by using a St Thomas cardioplegic solution (in mM): MgCl2 16, NaCl 147, KCl 20 and CaCl2 0.5. This solution was injected via the ascending vena cava with a peristaltic pump (10 ml/min) at a temperature of 4°C, until cardiac contractions stopped. The hearts were then excised and immersed in the ice cold cardioplegic solution. A cannula was put into the aorta and the coronary vessels were washed for 1 min with the solution (2 ml/min). Four conditions were investigated:

Cardioplegia: The hearts were used immediately for fiber preparation (time "0 h" of preservation).

Immersion: After cardioplegia, the hearts were kept in 20 ml of the ice cold cardioplegic solution (4°C) for 6 or 15 h (Immersion 6 h or 15 h).

Low-flow perfusion: The hearts, immersed in the ice-cold cardioplegic solution, were aortically perfused with the air-equilibrated cardioplegic solution (4°C), at a flow rate of 0.3 ml/min for 15 h (Perf. 15 h).

2.2 Evaluation of energy status and function
2.2.1 31P nuclear magnetic resonance (31P-NMR) spectroscopy experiments
At the end of cardioplegia or of the preservation period, the hearts were put into a glass chamber filled with ice-cold cardioplegic solution, which was continuously renewed by a peristaltic pump (10 ml/min). The chamber was introduced into a 5-turn solenoidal coil (15 mm long and 18 mm in internal diameter). The 31P-NMR spectra were collected in a vertical 4.7 T magnet (Oxford Instruments) connected to a Bruker CXP 200 spectrometer. Each spectrum (10 min) resulted from the accumulation of 160 signals provoked by 17 µs pulses applied at 3 s intervals. The areas under the inorganic phosphate (Pi) and ATP (β-phosphate group) peaks were measured by planimetry, and the [ATP]/[Pi] ratio was calculated as a bioenergetic index.

2.2.2 Reperfusion of the hearts and measurement of functional parameters
At the end of cardioplegia or of the preservation period, some hearts from each group were subjected to aortic reperfusion with a physiological solution (in mM): NaCl 129, KCl 5.6, MgCl2 2.4, CaCl2 1.5, NaHCO3 21, glucose 11. When equilibrated with a O2/CO2 mixture (95/5%), the solution had a pH of 7.4. The hearts were gradually rewarmed from 4°C to 37°C (2°C per 2 min). At the beginning of reperfusion, the perfusion pressure was equal to 30 cm H2O. When the temperature reached 20°C, it was progressively raised by 5 cm per 2 min. Within 36 min, the hearts were perfused at 37°C and under 80 cm H2O of perfusion pressure. The hearts were then paced at a constant frequency of 6 Hz (360 beats/min). At the end of 60 min of stabilisation, the left ventricular developed pressure was evaluated using an intraventricular latex balloon, introduced into the left ventricle and connected by a short (15 cm) water-filled catheter to a Gould Statham pressure transducer, interfaced with a Gould 8188-3302-OX recorder.

2.3 Skinned fibers
This method has been exhaustively described and discussed in earlier studies [15, 16].

Small pieces of cardiac muscle were taken from the middle of the left ventricle and put into cold (4°C) solution A. All procedures were carried out at 4°C. These samples were rapidly dissected into bundles of fibers. Fibers were incubated in 1.8 ml of solution A in the presence of saponin (50 µg/ml) in order to destroy selectively the sarcolemma, and were shaken for 30 min. The bundles were then put into solution B (twice for 10 min) to wash out adenine nucleotides, phosphocreatine, and saponin. Oxygraphic measurements were performed with solution B. For the test of intactness of the outer mitochondrial membrane a "KCl solution" was used.

Solutions A and B were prepared on the basis of the cytoplasmic composition of the muscle cells.

Solution A (in mM): CaK2EGTA 2.77, K2EGTA 7.23 (pCa=7), MgCl2 6.56, dithiothreitol (DTT) 0.5, K-methanS 50, imidazole 20, taurine 20, Na2ATP 5.3 and PCr 15, pH 7.1 adjusted at 25°C.

Solution B (in mM): CaK2EGTA 2.77, K2EGTA 7.23 (pCa=7), MgCl2 1.38, DTT 0.5, K-methanS 100, imidazole 20, taurine 20, K2HPO4 3 and pyruvate 5, pH 7.1 adjusted at 25°C.

In "KCl solution" the medium was enriched with KCl 125 mM. Under these conditions, the labile compounds of the respiratory chain, such as cytochrome c, were dissociated from the inner membrane.

"KCl solution" (in mM): KCl 125, HEPES 20, glutamate 4, malate 2, Mg acetate 3, KH2PO4 5, EGTA 0.4 and DTT 0.3, pH 7.1 adjusted at 25°C, and 2 mg of bovine serum albumin (BSA) per ml were added.

Dithiothreitol was added in all solutions for protecting sulfhydryl groups of mitochondrial proteins, mainly that of creatine kinase, against oxidation.

2.4 Oxygraphy
The respiratory rates of skinned fibers (approximately 5 mg) were determined using a Clark electrode in an oxygraphic cell containing 2 ml of solution B supplemented with BSA (2 mg/ml) or 2 ml of "KCl solution", at 25°C, with continuous stirring.

The solubility of oxygen was taken to be 430 ng atom.O/ml.

2.4.1 Test of intactness of the outer mitochondrial membrane in "KCL solution"
The initial rate of respiration in skinned cardiac fibers was measured in a "KCl solution" containing substrates and no ADP. Then the respiration was stimulated by the addition of ADP at a final concentration of 1 mM which induced a maximal activation of respiration. Cytochrome c was added at a final concentration of 8 µM to test the intactness of the outer membrane. When the outer membrane is intact, the endogenous cytochrome c stays in the intermembrane space and the addition of cytochrome c (in excess) has no effect on the respiratory rate. If the outer membrane is damaged, the endogenous cytochrome c leaves the intermembrane space and the addition of cytochrome c stimulates the respiratory rate.

The acceptor control ratio (ACR) was evaluated by the ratio of respiratory rate with substrates and ADP 1 mM to the value before addition of ADP.

2.4.2 Respiration variables
Dependence on ADP: The respiratory rate of mitochondria in skinned cardiac fibers was measured in solution B, and different amounts of ADP (in final concentrations from 0.0125 to 1 mM) were successively added. The stimulatory effect of ADP was calculated from the respiration rates measured in the presence of a given concentration of ADP minus the value in the absence of ADP ({Delta}V). The apparent Km for ADP (the ADP concentration which was necessary to obtain half the maximal activation) and the maximal stimulation of the respiratory rate induced by ADP ({Delta}Vmax) were calculated from double-reciprocal plots of the dependence of respiration rate ({Delta}V) on the concentration of ADP [14].

Dependence on ADP in presence of creatine: The apparent Km for ADP and {Delta}Vmax were determined in the presence of creatine (20 mM) in solution B, as previously described, in another series of measurements with different ADP concentrations.

2.4.3 Direct evaluation of the stimulating effect of creatine on respiration
The respiratory rates (V) of mitochondria in skinned cardiac fibers were measured in solution B. The initial respiration was stimulated by the addition of ADP to a non-saturating concentration of 0.1 mM (VADP), and creatine was added to a final concentration of 20 mM (VCr). This protocol aimed to evaluate directly the stimulatory effect of creatine from one oxygraphic experiment on a single fiber preparation. The (VCr–VADP)/VADP index which was calculated quantifies this effect.

At the end of the respiration determinations, the skinned fibers were washed in water and the dry weights were determined, in order to express the absolute values of respiration rates in ng atom O/min/mg dry weight (ng at.O/min/mg d.w.).

2.5 Statistical analyses
Values in the tables and figures are expressed by means±s.e.m. The apparent Km for ADP was estimated from a linear regression of double-reciprocal plots.

Statistical comparisons were made using the ANOVA test (variance analysis and Fischer's test), and P<0.05 was taken as the level of significance.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Energy state and mechanical activity of the rat heart after preservation
Fig. 1 shows that the 31P-NMR determined ATP/Pi ratio in the hearts decreased with time during preservation. This decline in ATP/Pi ratio was slowed by low-flow perfusion (0.3 ml/min) of cardioplegic solution during heart preservation.


Figure 1
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  		Fig. 1 Impact of the duration (0, 6, 15 and 24 h) and mode (immersion or low-flow perfusion) of heart preservation was evaluated by measuring a metabolic and a functional variable. The immersion mode is indicated by open symbols and the perfusion mode by closed symbols. The ATP/Pi ratio was calculated from the areas measured under inorganic phosphate and ATP (β-phosphate group) peaks of 31P-NMR spectra (10 min), at the end of preservation. Cardiac recovery was evaluated, at the end of the reperfusion (1 h 40), by measuring the left ventricular developed pressure (LVDP) using an intraventricular latex balloon. At time "0 h", the ATP/Pi ratio was evaluated immediately on isolated hearts after cardioplegia and the LVDP was determined on the reperfused cardioplegic hearts. The values are means±s.e.m. of (n) hearts; P<0.05 was taken as the level of significance. For comparison of LVDP all the groups were taken into account. When all the groups were taken into account, comparison of ATP/Pi ratio showed that the value obtained in the cardioplegic group was significantly higher than in the other groups. Differences between the various conditions of preservation were observed only when the high value of the ATP/Pi ratio in the cardioplegic group was excluded from comparison. *Different from all other groups. {dagger}Different from 6 h. {ddagger}Different from 15 h perfusion. §Difference between immersion and perfusion.

 
When reperfused with a physiological solution the hearts regained mechanical function (left ventricular developed pressure: LVDP) at a relatively stable level during a 60 min period of perfusion following stabilisation of temperature, heart rate and perfusion pressure. Functional recovery was all the lower as the duration of preservation became longer. As for the ATP/Pi ratio, low-flow perfusion during preservation allowed a better recovery of function than did simple immersion.

Since the ratio of ATP/Pi was affected to different degrees in the experiments described in Fig. 1, it was of importance to know the changes in mitochondrial function in preserved hearts under these conditions. Therefore, we decided to investigate 3 selected conditions of preservation. The first two included hearts submitted to 6 h of immersion and 15 h of perfusion. These hearts displayed an identical ATP/Pi ratio (equal to 0.25), but they showed a statistically different LVDP on reperfusion (respectively 65 and 40% of the control value). The third condition was 15 h of immersion, which induced extensive energy depletion and very poor mechanical recovery of the hearts.

3.2 Mitochondrial function
3.2.1 Tests of intactness of mitochondrial membranes
Firstly, we tested the intactness of mitochondrial membranes in the different groups.

Recordings in Fig. 2 illustrate the effect of the addition of 8 µM exogenous cytochrome c on the respiration induced by 1 mM ADP. The determinations were made in a KCl (125 mM) medium in which endogenous cytochrome c dissociates from the outer surface of the inner mitochondrial membrane [22]. Under these experimental conditions, when the outer membrane is intact, cytochrome c remains in the intermembrane space and maintains a high respiratory activity [23]. In this case, the addition of cytochrome c has no effect on the respiratory rate (Fig. 2a, trace from control heart). If the outer membrane is damaged, cytochrome c may leave the mitochondrion, and the addition of a high concentration of cytochrome c (8 µM) increases the respiratory rate (Fig. 2b, trace from ischemic heart).


Figure 2
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  		Fig. 2 Test of intactness of the outer mitochondrial membrane. The oxygraph traces show respiratory activities of mitochondria in skinned cardiac fibers in "KCl medium". At the time indicated, fibers, ADP (1 mM) and cytochrome c (Cyto. c) were added. The traces are representative of 5–7 experiments. In fibers from the control group (a) the addition of cytochrome c had no effect on respiration. In fibers from the ischemic group (b), stimulation of respiration by the addition of cytochrome c revealed a leak of endogenous cytochrome c through the altered outer mitochondrial membrane.

 
Fig. 3 shows these results for different groups of experiments with their statistical evaluation. In the fibers from hearts subjected to 1 h of ischemia at 37°C the value of respiration with 1 mM ADP was decreased (although not statistically different from control values), but the addition of cytochrome c significantly accelerated this respiratory rate. In these fibers the respiratory rate in the absence of ADP was significantly increased. These changes indicate the presence of alterations at the level of the outer membrane (effect of cytochrome c) and the inner membrane (increase of respiratory rate with only substrates). In this group, the acceptor control ratio (ACR: ratio of respiratory rate with substrates and ADP 1 mM to the value before ADP addition) was greatly decreased (2.9±0.4 versus 6.9±0.4 in controls) due to the increase in the initial respiratory rate (with only substrates), and the addition of cytochrome c did not completely correct this ACR value.


Figure 3
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  		Fig. 3 Respiratory parameters of skinned fibers in "KCl medium". This figure shows the results of oxygraphy measurements obtained for each group in the experiments described in Fig. 2. The oxygen consumption rates in the presence of only substrates (grey bars), in the presence of 1 mM ADP (hatched bars), and in the presence of 1 mM ADP + 8 µM cytochrome c (closed bars), are expressed in ng at. O/min/mg d.w. The different experimental groups are: cardioplegic hearts, time "0" of the preservation period (Immersion 0 h); hearts preserved by immersion for 6 or 15 h (Immersion 6 h or 15 h); hearts preserved by low-flow perfusion for 15 h (Perf. 15 h). The two additional groups for comparison are: hearts quickly removed without cardioplegia (Cont.); hearts subjected to 1 h of normothermic total ischemia (Isch.). The values are means±s.e.m. from 5–7 hearts; P<0.05 was taken as the level of significance. *Different from controls, immersion 0 and 6 h, and perfusion 15 h. {dagger}Different from immersion 15 h. {ddagger}Different from respiratory rate before addition of cytochrome c; comparison using a statistical test for repeated measurements within each group.

 
In fibers from hearts which were subjected to cardioplegia, all the values measured were identical to those evaluated in controls and the ACR value was equal to 6.8±0.6.

No difference was observed between the groups of hearts that were preserved for 6 h by immersion or 15 h by perfusion and the control or cardioplegic groups. The ACR values were respectively equal to 6.4±0.6 and 6.6±0.4.

On the contrary, in fibers from hearts preserved for 15 h by simple immersion, as observed in fibers from hearts subjected to normothermic ischemia, the addition of cytochrome c significantly increased the respiration of skinned fibers. The ACR value was significantly decreased (4.5±0.4) in comparison with the other groups, but it was restored to control values in the presence of cytochrome c. Thus, in these hearts some damage to the outer membrane had occurred whereas, as indicated by the unchanged value of initial respiratory rate with substrates and without ADP, the inner mitochondrial membrane seemed to remain intact.

3.2.2 Regulation of respiration of skinned cardiac fibers by ADP and creatine: determination of apparent Km for ADP
In the absence of creatine: In cardiac cells in vivo, the diffusion of ADP seems to be limited by the low permeability of the mitochondrial outer membrane to this substrate [24, 25]. It was thus of interest to study, in more detail, the alterations in the kinetics of regulation of respiration by ADP in fibers of preserved hearts. In good agreement with many earlier works [14, 15, 23–25], the respiration rate, which was measured on skinned fibers from control hearts, slowly increased with the increase in ADP concentration in the reaction medium. A maximal activation of respiration was obtained with millimolar ADP concentrations.

The results obtained from analysis of these dependences for each group are statistically analysed in Fig. 4A. In the cardioplegic group, the apparent Km for ADP was in the same range of values as in controls (275±27 versus 291±25 µM). The apparent Km for ADP was not significantly altered either in the fibers from hearts preserved under low-flow perfusion (252±39 µM) or in the fibers from hearts preserved for 6 h by immersion (235±36 µM).


Figure 4
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  		Fig. 4 Control of oxidative phosphorylation by ADP. (A) Determination of the apparent Km for ADP in the presence or absence of creatine. The apparent Km, expressed in µM, was calculated from double-reciprocal plots of the dependence of the ADP stimulated respiration rate ({Delta}VO2) on the concentration of ADP, in the presence (hatched bars) or absence (grey bars) of creatine (20 mM). (B) Determination of maximal stimulation of the respiratory rate induced by ADP ({Delta}Vmax), in the presence or absence of creatine. The {Delta}Vmax, expressed in ng at. O/min/mg d.w., was calculated from double-reciprocal plots of the dependence of the ADP-stimulated respiration rate ({Delta}VO2) on the concentration of ADP, in the presence (hatched bars) or absence (grey bars) of creatine (20 mM). The values are means±s.e.m. from 5–7 hearts; P<0.05 was taken as the level of significance. For description of experimental groups, see legend to Fig. 3. *Different from controls and immersion 0 h (cardioplegia). {dagger}Difference between immersion 15 h and perfusion 15 h. {ddagger}Different from value determined without creatine in the medium. §Different from immersion 0 h.

 
On the contrary, in the fibers from hearts preserved for 15 h by simple immersion, the apparent Km for ADP was significantly decreased (to 156±28 µM), as well as in the fibers from hearts submitted to 1 hour of normothermic ischemia (117±17 µM).

The addition of cytochrome c to the respiration medium (solution B), which corrected the loss of this compound when the outer membrane was damaged, did not modify the value of the apparent Km (results not shown).

In the presence of creatine: Because of the presence of the specific isoenzyme of creatine kinase in the mitochondria of cardiac cells, addition of creatine stimulates production of ADP in the intermembrane space and in this way stimulates respiration [14]. This effect of creatine on respiration is decreased if creatine kinase dissociates from the mitochondrial membrane [26–28].

The effect of the addition of 20 mM creatine on the kinetics showing the response to increasing concentrations of ADP is statistically analysed in Fig. 4 A. In the control group, the apparent Km for ADP value was significantly decreased from 291±25 µM in the absence of creatine to 90±12 µM in its presence.

In the fibers from cardioplegic or preserved hearts, the apparent Km observed in the presence of creatine was in range of control values (88±8 µM). It was slightly, but not significantly, increased in the fibers from normothermic ischemic hearts (116±27 µM).

Fibers isolated from hearts preserved for 6 h by immersion or for 15 h by low-flow perfusion displayed very similar characteristics compared with control and cardioplegic groups: the apparent Km for ADP was in range of 250–240 µM in the absence of creatine and 80–90 µM in the presence of creatine.

Thus, when the results obtained in the presence or in the absence of creatine are statistically analysed, the effect of creatine on apparent Km of ADP appears unchanged in these two groups of preserved hearts (Imm. 6 h and Perf. 15 h). In the fibers from hearts preserved for 15 h without perfusion, as the apparent Km for ADP in the absence of creatine was significantly decreased, the difference with and without creatine becomes statistically non-significant. In the totally ischemic hearts, the effect of creatine on the apparent Km for ADP was completely lost.

3.2.3 Determination of the maximal respiration activated by ADP
The {Delta}Vmax (see Section 2) determined from double reciprocal plots represents the maximal respiratory rate induced by ADP. The results are shown in Fig. 4B. The values obtained in the fibers from control and cardioplegic hearts were in the range of 35 ng at.O/min/mg d.w. These values are close to the respiratory rates of isolated mitochondria when calculated relative to cytochrome aa3 [13].

In fibers from normothermic ischemic hearts, the {Delta}Vmax with or without creatine represented no more than 40% of the control values, suggesting extensive alterations in mitochondrial function.

In fibers from hearts preserved for 6 h by immersion no significant change occurred.

In fibers from hearts preserved 15 h by simple immersion, the {Delta}Vmax, measured in the absence and in the presence of creatine, was significantly decreased by approximately 40%.

The low-flow perfusion of hearts during preservation restored the {Delta}Vmax determined in absence of creatine, but not completely the {Delta}Vmax determined in presence of creatine.

It should be noted that the addition of creatine (20 mM) slightly increased the maximal respiratory rate induced by ADP. This increase of {Delta}Vmax in the presence of creatine was not statistically significant except in the fibers from 6 h preserved hearts (smaller scatter of individual values).

3.2.4 Direct evaluation of the stimulating effect of creatine on respiration
As illustrated in Fig. 5, the stimulating effect of creatine on mitochondrial respiration could also be investigated directly by evaluating the respiration rate at a non-saturating concentration of ADP (0.1 mM). This simple test allowed evaluation of the stimulating effect of creatine directly from a single oxygraphic experiment. The index, which was calculated from the respiratory rates measured before and after the addition of creatine to the medium [(VCr–VADP)/VADP], decreased with the extent of ischemic damage (Fig. 6): from a non-significant decrease in fibers from hearts preserved for 6 h by immersion up to more than 85% in fibers from hearts subjected to 1 h of normothermic ischemia or 15 h of preservation by immersion.


Figure 5
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  		Fig. 5 Stimulation of respiratory rate by creatine. The oxygraph traces show respiratory activities of mitochondria in skinned cardiac fibers in "solution B" (see Section 2) when, at the indicated time, fibers, ADP (0.1 mM) and creatine (20 mM) were added. The traces are representative of 5–7 experiments. In fibers from the control group (a) the addition of creatine stimulates respiration. In fibers from the ischemic group (b), no stimulation of respiration by the addition of creatine was detected.

 

Figure 6
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  		Fig. 6 Degree of activation by creatine of the respiration in skinned cardiac fibers. This figure shows the results of oxygraph measurements obtained for each group in the experiments described in Fig. 5. The (VCr–VADP)/VADP ratio shows the degree of activation of respiration by creatine 20 mM, at ADP 0.1 mM. VADP is the respiration rate at 0.1 mM ADP in the absence of creatine. VCr is evaluated with 0.1 mM ADP + 20 mM creatine. Values are means±s.e.m. from 5–7 hearts; P<0.05 was taken as the level of significance. For description of experimental groups, see legend to Fig. 3. *Different from controls and immersion 0 h (cardioplegia). {dagger}Difference between immersion 15 h and perfusion 15 h.

 
This determination revealed a slight alteration in the stimulatory effect of creatine in the fibers from hearts preserved for 15 h by low-flow perfusion, in which the apparent Km for ADP was not significantly altered. Therefore, the index (VCr–VADP)/VADP seems to be the most sensitive indicator of early ischemic damage.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The skinned fiber technique which we used in this study is particularly suited to the goal of this investigation for the following reasons: (1) the whole mitochondrial population of the cell is accessible for measurements, and mitochondria are studied in their natural surroundings; (2) the isolation artefacts are excluded—this is especially important since several properties of mitochondria, such as sensitivity to ADP, are significantly modified during isolation [24]; and (3) this technique allows evaluation of the functional coupling of mitochondrial creatine kinase to other mitochondrial enzymatic systems. In addition, this technique can also be used in clinical situations since it requires only a very small piece of tissue (5–10 mg). This technique has already been effectively applied in studies of mitochondria in normal and pathological cardiac and skeletal muscles [13–18], including primary genetically induced mitochondrial diseases and dilated cardiomyopathy in patients [19]. The advantages and limitations of this technique have been discussed previously in these papers.

When the myocardial tissue is irreversibly injured, such as after 1 h normothermic (37°C) ischemia, the permeabilized fiber technique reveals: (1) damage to the outer mitochondrial membrane (effect of cytochrome c); (2) damage to the inner mitochondrial membrane (increase in initial respiratory rate with only substrates); (3) decrease in {Delta}Vmax; (4) loss of the stimulating effect of creatine.

After preservation, only the hearts preserved for 15 h by simple immersion showed similar severe disturbances in mitochondrial structure and function.

Under the other conditions of preservation (6 h immersion, 15 h under low-flow perfusion), damage to mitochondrial function was far less severe, and a gradation of the severity of these alterations can be seen from the experimental results. Indeed, following 6 h of preservation by simple immersion, no significant change occurred in all parameters. After 15 h of preservation with low-flow perfusion, only a slight decrease in {Delta}Vmax measured in the presence of creatine and a fall in the stimulating effect of creatine were observed. These results suggest that the respiratory chain function is not deeply affected under these conditions. This is in accordance with the observation by Piper et al. [2, 3] that this kind of alteration appears later than functional damage. Our observations lead to the conclusion that the earliest damage to the mitochondria during long-term hypothermic ischemia occurs at the level of the outer mitochondrial membrane and of the intermembrane space.

Recent studies of the kinetics of respiratory regulation by ADP in skinned fibers have suggested that the permeability of the outer mitochondrial membrane to ADP is limited due to some cytoplasmic protein factor attached to this membrane which may control the activity of porin channels [24, 25]. The coupled creatine kinase overcomes this effect by increasing the turnover of adenine nucleotides in the intermembrane space of mitochondria. The beneficial effects of coupled creatine kinase producing ADP in the vicinity of translocase can also be perceptible from the {Delta}Vmax value which is always systematically higher in the presence of creatine. This observation can be explained by a local production of protons in the creatine kinase reaction, thus maintaining the electrogenic exchange of adenine nucleotides by translocase.

The decrease in the apparent Km for ADP (in the absence of creatine) that we have observed during heart preservation can result both from dissociation of the hypothetical protein factor that controls porin permeability for ADP and/or from some rupture in the outer mitochondrial membrane. As a consequence, one could expect also a decrease in the apparent Km for ADP in the presence of creatine. Indeed, the value of the apparent Km observed in fibers from the control group (in the presence of creatine) is 4 times higher than that observed in isolated mitochondria (80 versus 20 µM) in which the addition of creatine has no effect [19]. Therefore, if outer membrane permeability is increased while creatine kinase remains coupled, one can expect that the apparent Km for ADP measured in presence of creatine would be lower than 80 µM. This is not what we observed in fibers from preserved hearts since the Km value remained in the range of 80 µM. We can therefore hypothesize that mitochondrial creatine kinase is partially decoupled from adenine nucleotide translocase. The loss of the stimulatory effect of creatine, measured at fixed low unsaturated ADP concentration (0.1 mM) in fibers from immersed hearts, confirms this hypothesis. A decrease of this effect was also observed in fibers from hearts preserved by perfusion in the absence of any change in apparent Km. This lower degree of stimulation of respiration by creatine can be explained by the slight decrease in {Delta}Vmax observed in these hearts, since the creatine-stimulated respiration rates at a fixed ADP concentration are influenced also by {Delta}Vmax. The physico-chemical conditions for the determination of mitochondrial function in permeabilized fibers are obviously not identical to those prevailing in the ischemic cell (concentration of Pi, Ca2+ etc.). It cannot be excluded that some ischemia-induced alterations could be corrected by the experimental procedure itself. Thus, the addition of dithiothreitol to the solutions might attenuate or reverse some possible damage to creatine kinase caused by reactive oxygen species [29, 30]. We therefore assume in the following discussion that the alterations we detect are the durable ones (not necessarily irreversible) that cannot be reversed by the experimental procedure.

From our results, we can suggest that some defect in energy transport (i.e., that of the functional coupling of mitochondrial creatine kinase: CKm) may have occurred to a different extent depending on the method and duration of preservation. These observations are in good agreement with those of Bittl et al. [31, 32] who showed that: (1) in the post-ischemic rat heart, a close correlation exists between the reduction in performance and the decline in CKm activity [31] and (2) the decrease in the turnover rate of high-energy compounds through the CK reaction may contribute to the metabolic basis for contractile failure during hypoxia [32]. The important role of energy channelling in mechanical activity is also stressed by recent studies in which a reduction in CK activity, while not limiting the baseline contractile performance, largely impairs the mechanical response to inotropic stimulation [33] or the restoration of mechanical activity after acute hypoxia [34]. Also the results of a detailed analysis of metabolic changes in different cellular compartments of ischemic cardiac cells have allowed Schultheis's group [35] to conclude that a decoupling between creatine kinase and translocase may contribute significantly to early cardiac failure. This occurs by decreasing the energy flux via the phosphocreatine pathway to myofibrils, resulting in a local elevation of ADP in myofibrils and an inhibition of crossbridge sliding [36, 37]. Such a decoupling between the phosphocreatine shuttle and the ADP/ATP carrier might reduce the high-energy phosphate supply to myofibrils and could contribute substantially to the processes leading to ischemic failure. The loss of creatine kinase–adenine nucleotide translocase coupling may result from the dissociation of mitochondrial creatine kinase from the external face of the inner mitochondrial membrane. In fact, it is essential that creatine kinase is attached to the inner membrane in the vicinity of translocase in order to control respiration by direct substrate-product channelling [22–25, 27]. It has been shown in several laboratories that the experimental detachment of creatine kinase from the membrane practically eliminates this kind of control of respiration [26–28, 38].

Thus the question arises: What are the mechanisms responsible for these effects on creatine kinase?

It is known, from the first morphological studies of ischemic myocardium, that mitochondrial swelling occurs very rapidly, within the first 15 min of ischemia, even in the case of reversible ischemic damage [1, 2], due to an accumulation of inorganic phosphate. This swelling should result in some rupture of the outer mitochondrial membrane [1]. Alternatively, Vial et al. [26] and recently Soboll et al. [38] and Veksler and Ventura-Clapier [28] proved that for isolated mitochondria and skinned cardiac fibers, inorganic phosphate in concentrations of 10–20 mM causes a dissociation of creatine kinase from the inner mitochondrial membrane, especially if the rise in inorganic phosphate precedes the intracellular ischemia-induced acidification. Veksler and Ventura-Clapier [28] showed that this dissociation can be reversed by reperfusion after a short period of ischemia when the phosphate concentration returns to its low value. Earlier, DeLuca and Hall had established that phosphate decreased the creatine-stimulated respiration of isolated heart mitochondria [39]. This can occur even if the outer membrane is not damaged; indeed, the dissociation of creatine kinase from the inner membrane eliminates completely the functional coupling between creatine kinase and adenine nucleotide translocase [27]. When considering the overall results of this study, we observe that the mechanical activity and the stimulating effect of creatine, expressed by the ratio "(VCr–VADP)/VADP", decrease in parallel when the duration of preservation or the severity of ischemia increases, while Pi accumulates (ATP/Pi ratio decrease). Thus, the accumulation of Pi during heart preservation could participate in the development of the mitochondrial swelling observed in electron microscopy and solubilization of creatine kinase. It can also play a major role at the level of the myofibrils. Indeed, many authors report that Pi, in a range of 1–30 mM, reduces the maximum Ca2+-regulated force and shifts the sigmoidal relationship between force and calcium to higher concentrations [40, 41]. Some other factors such as protons, accumulation of ADP and reduction of phosphocreatine concentration can also contribute significantly to the reduction of force generation [40, 41].

In conclusion, our experiments demonstrate that, during long-term preservation of the rat heart, alterations to the process of channelling of energy from mitochondria occur before any other damage to mitochondrial respiratory function can be seen. This probably results from mild alterations at the level of the permeability for ADP of the outer mitochondrial membrane and of the creatine kinase coupled reaction in the intermembrane space even in the absence of rupture of the membrane. We hypothesize that the accumulation of inorganic phosphate in the cell that occurs during ischemia could be responsible for these alterations and for the consequent decrease of function.

In addition, the results of this study show that the low-flow perfusion of the heart during preservation, which results in better cardiac recovery and reduces Pi accumulation, helps to protect mitochondrial function.

Finally, the results of this study show that the assessment of mitochondrial parameters sensitive to organelle swelling—intactness of the outer membrane, and creatine-stimulating effect—may be a useful and sensitive additional tool for the evaluation of early hypoxic or ischemic damage to mitochondria. Determination of these parameters in biopsy samples by using the skinned fiber technique could be used as a simple and rapid test for evaluating the efficiency of the protective measures in heart surgery.

Time for primary review 31 days.


   Acknowledgements
 
The authors thank Dr. Renee Ventura-Clapier for a critical reading of this manuscript. V.A. Saks's position of invited professor in the laboratory, part-time, is supported by the French Ministry of Higher Education and Research. Part of this study was supported also by an INSERM grant (No. 94 EW 10), by a grant from the Region Rhône-Alpes and by INTAS grant No. 94 4738.


   Notes
 
1 On leave from Laboratory of Bioenergetics, Institute of Chemical and Biological Physics, Tallinn, Estonia. Back


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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