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Cardiovascular Research 1999 43(2):408-416; doi:10.1016/S0008-6363(99)00100-5
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Pretreatment with PKC activator protects cardiomyocytes against reoxygenation-induced hypercontracture independently of Ca2+ overload

Yury V. Ladilov, Claudia Balser-Schäfer, Steffen Haffner, Hagen Maxeiner and H.Michael Piper*

Physiologisches Institut, Justus-Liebig-Universität, Aulweg 129, D-35392 Giessen, Germany

* Corresponding author. Tel.: +49-641-994-7241; fax: +49-641-994-7239 michael.piper{at}physiologie.med.uni-giessen.de

Received 7 September 1998; accepted 12 February 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusion
 Acknowledgments
 References
 
Objective: Although several studies have shown that activation of protein kinase C (PKC) plays an important role in protection through ischemic preconditioning, little is known about the effects of direct PKC activation on the course of ischemia-reperfusion injury. The aim of this study was to analyse the effects of a pretreatment with the PKC activator 1,2-dioctanoyl-sn-glycerol (1,2DOG). Methods: Isolated adult Wistar rat cardiomyocytes were exposed to 80 min of simulated ischemia (anoxia, pHo6.4) and 20 min of reoxygenation (pHo7.4). Cytosolic Ca2+ (fura-2), cytosolic pH (BCECF), Mg2+ (Mg-fura-2), lactate and cell length were measured and compared between control cells and cells treated with 20 µmol/l 1,2DOG before anoxia (10 min treatment and 10 min wash out). Results: 1,2DOG-pretreatment delayed the time to extreme ATP depletion, but had no effect on lactate production and cytosolic pH. The accumulation of cytosolic Ca2+ was markedly accelerated in pretreated cells that developed rigor shortening, but reoxygenation-induced hypercontracture was significantly reduced. 1,2DOG, therefore, completely abolished Ca2+-dependence of hypercontracture. The effects of pretreatment were fully abolished with 1 µmol/l bisindolylmaleimide (PKC inhibitor). We conclude that PKC preactivation leads to (1) reduction of energy demand, (2) acceleration of Ca2+ overload during anoxia and (3) prevention of reoxygenation-induced hypercontracture independent of anoxic changes in cytosolic Ca2+ and pH.

KEYWORDS Calcium (cellular); Ischemia; Preconditioning; Protein kinases; Reperfusion

See Editorial of this article by D.F. Stowe (pages 285–287) in this issue.


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 5 Conclusion
 Acknowledgments
 References
 
Activation of protein kinase C (PKC) has been shown to be an important part of the protective mechanism of ischemic preconditioning [1,2]. In the whole heart the beneficial effects of ischemic preconditioning can be imitated by direct activation of PKC with diacylglycerols before ischemia [2–4]. Similarly, activation of PKC in single cardiomyocytes also provided significant protection [5,6]. However, the detailed cellular mechanisms of protection through PKC preactivation are still poorly understood.

In the present study we used the model of isolated rat cardiomyocytes exposed to simulated ischemia (anoxia, extracellular pH (pHo) 6.4) and reperfusion (reoxygenation, pHo 7.4) which was characterised previously [7]. Depleted of oxygen and substrates during ischemia, cardiomyocytes rapidly lose ATP, and then accumulate Ca2+ in the cytosol [7–9]. It has been shown in previous studies that the combination of excessive cytosolic Ca2+ overload with the resupply of energy during reperfusion leads to hyperactivation of myofibrils and hypercontracture of cardiomyocytes [10,11]. In tissue, hypercontracture causes membrane disruption and cell death as a result of cell-to-cell force transduction [12]. Therefore, hypercontracture of cardiomyocytes promoted by excessive accumulation of Ca2+ represents an important element of reperfusion-induced injury. Reoxygenation-induced hypercontracture has been previously found to be reduced after a single PKC preactivation [6]. In the present study the cellular mechanism of this protection was analysed. It was investigated whether PKC preactivation protects against reoxygenation-induced hypercontracture due to (1) a delay of anoxic ATP depletion, (2) a reduction of anoxic Ca2+ overload or (3) altered control of pHi during anoxia and/or reoxygenation.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 5 Conclusion
 Acknowledgments
 References
 
2.1 Preparation of isolated cardiomyocytes
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 1996).

Ventricular heart muscle cells were isolated from 200–250 g adult male Wistar rats, plated in medium 199 with 4% fetal calf serum on glass cover-slips which had been preincubated overnight with 4% fetal calf serum [13]. Four hours after plating, the cover-slips were washed with medium 199. As a result of the wash, broken cells were removed, leaving a homogeneous population (>95%) of rod-shaped quiescent cardiomyocytes attached to the cover-slip. From each isolation 2–3 cover-slips were used. On each cover-slip from four to six cells were investigated. Only cells exhibiting a rod shaped morphology and no signs of sarcolemmal blebbing were used for the experiments. These cells were found to have a low resting cytosolic Ca2+ concentration [7].

2.2 Ca2+, Mg2+, pH and cell length measurements
To measure cytosolic Ca2+, Mg2+ or H+ concentrations, cardiomyocytes were loaded in medium 199 at 35°C for 30 min with acetoxymethyl esters of fura-2 (2,5 µmol/l), Mg-fura-2 (1,5 µmol/l) or 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) (1,5 µmol/l), respectively. After loading, cells were washed twice with medium 199 following incubation in medium 199 for 30 min to allow hydrolysis of the acetoxymethyl esters within the cell. The fluorescence from dye-loaded cells was 20–30 times higher than background fluorescence from unloaded cells.

The cover-slip with loaded cells was introduced into a gas-tight, temperature-controlled (37°C), transparent perfusion chamber positioned in the light path of an inverted microscope (Diaphot TMD, Nikon, Düsseldorf, Germany). Alternating excitation of the fluorescent dye at wavelengths of 340 and 380 nm for fura-2, 340 and 375 for Mg-fura-2 or 450 and 490 nm for BCECF was performed with an AR-Cation Measurement System adapted to the microscope (Spex Industries, Grasbrunn, Germany). Emitted light (490–510 nm for fura-2 and Mg-fura-2 and 520–560 nm for BCECF) from a 10x10 mm area within a single fluorescent cell was collected by the photomultiplier of the Spex system. The light signal was recorded and analysed by an IBM PC/AT-based data analysis system (Model DM3000CM, Spex Industries, Grasbrunn, Germany).

In some experiments the maximal rate of the fura-2 ratio rise after rigor contracture was calculated. For this, the phase with the fura-2 ratio rising was divided in 2 min intervals and the increase in the ratio was determined for each interval. The maximal increase was expressed as d(fura-2 ratio)/dt.

Simultaneous with measurement of the fluorescence, the cell’s microscopic image was recorded with a video camera and stored on tape. From these recordings, changes of the cell length were determined later. In the case of hypercontracted cells, the cell dimension along its previous longitudinal axis was determined. The value of cell hypercontracture was expressed as a reduction of cell length during 20 min reoxygenation as percentage of cell length before anoxia:

Formula
where LA80 – cell length after 80 min of anoxia; LR20 – cell length after 20 min of reoxygenation, LN – cell length before anoxia.

2.3 Dye compartmentation and in vivo calibration
The loading protocols used were selected from a number of variations because they provided the highest yield in fluorescence and minimal dye compartmentation. To assess the extent of intracellular dye compartmentation, cells were chemically ‘skinned’ with digitonin as described previously [6]. This test showed that the fluorescent signal from intracellular stores did not exceed 10% for fura-2, 15% for Mg-fura-2 and 12% for BCECF compared with the signal from whole cells. Furthermore, the extent of dye compartmentation did not differ significantly between control cells and cells after anoxia and reoxygenation. For the purpose of the present study, therefore, correction of the data for this small extent of dye compartmentation seemed unnecessary.

Because of the inherent problems with calibration of fura-2 and Mg-fura-2 ratios, data were expressed in arbitrary units of fluorescence ratio. In vivo calibration of the BCECF ratio was performed according to Koop and Piper [14], with 10 µg/ml nigericin and various pH values.

2.4 L-Lactate measurement
To measure production of L-lactate, cells were plated in 35 mm diameter Petri dishes. After washing with anoxic Tyrode’s solution (pH=6.4), the dishes were placed into a temperature controlled (37°C) plexiglass chamber flushed with 100% nitrogen. Experiments were terminated after 90 min of anoxia by adding 0.5 ml of 1.2 mol/l HClO4 per dish to the culture medium. Protein was determined according to Bradford [15] using bovine serum albumin as standard. After neutralisation, perchloric acid extracts of cultures were analysed for L-lactate (L-lactate test kit, Sigma Chemie Co).

2.5 Media
The perfusion chamber (0.5 ml filling volume) was perfused at a flow rate of 0.6 ml/min with modified Tyrode’s solution containing (in mmol/l): 135.0 NaCl, 2.6 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.0 CaCl2, 25.0 HEPES and 5.0 glucose; pH was 7.4 at 37°C. Normoxic solution was equilibrated with air. The solution was made anoxic by autoclaving as described previously [16] and was equilibrated before and during use with 100% N2. Anoxic solution was glucose free and had pH=6.4.

2.6 Experimental protocols
Five sets of experiments were performed. In all of them, cells were first exposed to 20 min of normoxia at pHo 7.4, followed by 80 min of anoxia at pHo6.4 (simulated ischemia) and 20 min of reoxygenation at pHo7.4 (simulated reperfusion). In the control experiments (protocol A) this standard protocol was performed without modification. In protocol B, the first normoxic superfusion of cells was started with 1,2DOG (20 µmol/l) present for the first 10 min. It was washed out for the subsequent 10 min. In protocol C, cardiomyocytes were treated before anoxia with PKC-inactive 1,3DOG. In protocols D and E, cells were treated similarly as in protocols A and B with the addition of 1 µmol/l of PKC inhibitor bisindolylmaleimide 1 (BIM) during 20 min normoxia and subsequent 80 min anoxia.

2.7 Materials
Medium 199 was purchased from Boehringer-Mannheim, fetal calf serum from GIBCO, acetoxymethyl esters of fura-2, Mg-fura-2 and BCECF from Paesel and Lorey, 1,2- and 1,3-dioctanoyl-sn-glycerols from Sigma Chemical Co, bisindolylmaleimide 1 from Calbiochem-Novabiochem. All other chemicals were from Merck and of the highest purity available.

2.8 Statistics
Data are given as mean values±SE. For each experimental protocol more than 20 individual cells were used, with not more than six cells from the same cell isolates. Statistical comparisons were performed by analysis of variance (ANOVA) and use of the Bonferroni test [17]. Statistical significance was accepted when P<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 5 Conclusion
 Acknowledgments
 References
 
3.1 Influence of treatment with 1,2DOG on the time of rigor contracture and cytosolic Mg2+ concentration during anoxia
As shown before [18], during anoxia cardiomyocytes shorten eventually when the stores of ATP are depleted. This shortening is due to a rigor mechanism (rigor contracture). It is a rapid process of cell length reduction by about one third. Rigor contracture occurred under control conditions after 30.7±1.1 min of anoxia (Fig. 1). After pretreatment with 1,2DOG, the onset of rigor was significantly delayed. In 31% of the cells, rigor contracture no longer occurred within the investigated 80 min of anoxia. For the 69% of pretreated cells exhibiting rigor contracture within this period of anoxia, the average time of the onset of rigor contracture was delayed to 46.0±2.6 min. Pretreatment with PKC-inactive 1,3DOG had no effect on the mean time of rigor contracture. Nevertheless, variability of rigor time under this treatment was slightly reduced.


Figure 1
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  		Fig. 1 Histograms of the time of the onset of rigor contracture in control cells (n=79), cells pretreated with 1,2DOG (n=51) and cells pretreated with 1,3DOG (n=23). Data are presented as % of the total number of cells.

 
The fluorescent indicator Mg-fura-2 was used to monitor the rise in cytosolic free Mg2+ in anoxic cardiomyocytes. It has been shown previously that the concentration of Mg2+ in the cytosol increases during ischemia as a result of ATP degradation [19]. We found that the Mg2+ concentration, indicated by the Mg-fura-2 ratio, started to rise during anoxia just a few minutes before rigor contracture and reached its plateau shortly after completion of rigor contracture (Fig. 2). The time of the onset of rigor contracture and time of the end of Mg-fura-2 rise were, respectively, 28.9±2.1 and 29.3±2.7 min for protocol A (n=9), 50.2±3.9 and 50.7±3.1 min for protocol B (n=6) and 32.2±3.6 and 32.6±4.0 min (n=5) for protocol E.


Figure 2
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  		Fig. 2 An example of the temporal relationship between Mg-fura-2 ratio (original recording, arbitrary units) and cell length (–o–, % of initial length) in a control cardiomyocyte during anoxia. Mg-fura-2 ratio rises after 25 min of anoxia, indicating a rise of the cytosolic Mg2+ concentration. Five minutes later the cell undergoes rigor contracture and the Mg-fura-2 ratio levels off.

 
Delay of rigor contracture and of rise of the Mg-fura-2 ratio indicate that PKC preactivation exerts an energy-sparing effect. To analyse the cause of delayed ATP depletion in 1,2DOG-pretreated cells, L-lactate production during anoxia was measured. No difference was observed in L-lactate contents between control and 1,2DOG-pretreated cells after 90 min of anoxia (µg/mg protein: 8.0±0.5, n=8, and 7.2±0.6, n=7, respectively, P>0.05).

3.2 Influence of pretreatment with 1,2DOG on cytosolic Ca2+ concentration during anoxia and reoxygenation
During simulated ischemia cells accumulated Ca2+ in the cytosol, as indicated by a rise in the fura-2 ratio (Fig. 6). The accumulation of Ca2+ started only when the cells had developed rigor contracture, under all experimental conditions. Since the time of onset of rigor contracture became variable after 1,2DOG-pretreatment, the cells were separated in five groups for further analysis. The first group contained cells with rigor times less than 20 min of anoxia (control, n=10; 1,2DOG, n=0), the second group – cells with rigor times between 20 and 40 min (control, n=54; 1,2DOG, n=12), the third group – cells with rigor times between 40 and 60 min (control, n=15; 1,2DOG, n=16), the fourth group – cells with rigor times between 60 and 80 min (control, n=0; 1,2DOG, n=7) and the fifth group – cells which did not develop rigor contracture during 80 min of anoxia (control, n=0; 1,2DOG, n=16).


Figure 6
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  		Fig. 6 Temporal relationship between changes in the fura-2 ratio (continuous traces of original recordings, arbitrary units) and length (% of initial length) in a control cardiomyocyte (–o–) and in a 1,2DOG-pretreated cardiomyocyte (–{bigtriangleup}–) during anoxia and reoxygenation. Rigor contracture occurred at 41 min of anoxia for the control cell and at 43 min of anoxia for the 1,2DOG-pretreated cell. Upon reoxygenation, the control cell underwent hypercontracture, while the 1,2DOG-pretreated cell did not.

 
Under either experimental condition the end-anoxic level of the fura-2 ratio declined with the rigor time (Fig. 3). Control cells which developed rigor contracture between 40 and 60 min (group 3) had a significantly lower end-anoxic fura-2 ratio than cells in group 1. Pretreatment with 1,2DOG significantly enhanced cytosolic Ca2+ overload at the end of anoxia in cells from groups 2 and 3, as compared to control cells from similar groups. This corresponded to a more rapid rise of the fura-2 ratio in cells pretreated with 1,2DOG in which rigor contracture developed during the 20–40 and 40–60 min of anoxia (Fig. 4). In 1,2DOG-pretreated cells with rigor times between 60 and 80 min of anoxia (group 4) the end-anoxic cytosolic Ca2+ overload was significantly lower than in cells of groups 2 and 3. This may be because the time between onset of the Ca2+ overload (rigor contracture) and reoxygenation was very short in these cells (less than 20 min). In group 5, cardiomyocytes did not develop rigor contracture during 80 min of anoxia and exhibited a cytosolic Ca2+ level at the end of anoxia similar to that found under normoxic control conditions.


Figure 3
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  		Fig. 3 The fura-2 ratio (arbitrary units) at the end of 80 min anoxia in control cells (–o–) and cells pretreated with 1,2DOG (–bullet–). Broken line indicates the average level of the fura-2 ratio in normoxic cells. Data are means±SE. *, P<0.01 vs. corresponding control values. #, P<0.05 vs. control cells of group 1. §, P<0.05 vs. 1,2DOG-pretreated cells of groups 2 and 3.

 

Figure 4
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  		Fig. 4 The maximal rate of rise of the fura-2 ratio after anoxic rigor contracture in control cells (open bars) and 1,2DOG-pretreated cells (black bars). Data are means±SE. *, P<0.05 vs. corresponding control values.

 
During reoxygenation the fura-2 ratio fell rapidly to the initial value within 10 min (Fig. 6). The recovery of the fura-2 ratio was not significantly different between the groups (Table 2).


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  		Table 2 Comparison of effects of 1,2DOG, 1,3DOG and bisindolylmaleimide 1 (BIM)a

 
3.3 Protection of cardiomyocytes by pretreatment with 1,2DOG against reoxygenation-induced hypercontracture
During anoxia the cell length was reduced by rigor shortening by about 30% of the initial cell length under all experimental conditions. Reoxygenation of cells in the control group led to additional, irreversible reduction of cell length by about 30% of the initial cell length within first 5–6 min, i.e. hypercontracture (Fig. 5). In control cells, the extent of reoxygenation-induced hypercontracture was the same for all cells, irrespective of rigor time. 1,2DOG-pretreated cells with rigor time between 20 and 40 min of anoxia (group 2) developed the same degree of hypercontracture. In 1,2DOG-pretreated cells, developing rigor contracture after 40 min of anoxia or later (groups 3–5), reoxygenation-induced hypercontracture was greatly reduced.


Figure 5
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  		Fig. 5 Degree of hypercontracture (% of cell shortening relative to initial length) at the end of 20 min reoxygenation in control cells (–o–) and 1,2DOG-pretreated cells (–bullet–). Data are means±SE. *, P<0.05 vs. corresponding control values. §, P<0.05 vs. 1,2DOG-pretreated cells of group 2.

 
The comparison between control and pretreated cells in group 3 (Fig. 6) showed that in this group the pretreated cells exhibited much less hypercontracture than the control cells (Fig. 5). In the same group, however, the end-anoxic Ca2+ overload of pretreated cells exceeded that of control cells (Fig. 3). This finding indicates that protection against hypercontracture after 1,2DOG-pretreatment in this group is independent of the degree of Ca2+ overload at the end of anoxia. Indeed, the values of the end-anoxic fura-2 ratio and of hypercontracture were not correlated for pretreated cells of group 3, whereas they were correlated for control cells of group 3 (Fig. 7).


Figure 7
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  		Fig. 7 Relationship between the fura-2 ratio (arbitrary units) at the end of anoxia and hypercontracture (% of cell shortening relative to initial length) developed after 20 min of reoxygenation in control cells (o, n=15) and 1,2DOG-pretreated cells (bullet, n=16) of group 3. Degree of hypercontracture and fura-2 ratio at the end of anoxia are correlated in control cells (r=0.90, P<0.05). No correlation was found after 1,2DOG-pretreatment (r=0.002). Solid lines represent the regression lines for control and 1,2DOG-pretreated cells.

 
3.4 Changes of cytosolic pH during anoxia and reoxygenation
As described previously [7], pHi declined in cardiomyocytes exposed to anoxic media with pHo 6.4 and rapidly recovered to the initial level during reoxygenation with pHo 7.4. Interventions which delay the recovery of pHi during reoxygenation have been shown to protect against hypercontracture [7,20]. It was studied whether reduction of reoxygenation-induced hypercontracture in 1,2DOG-pretreated cells, especially cells of group 3, results from differences in pHi. We found that the pretreatment of the cells with 1,2DOG did not influence the development of cytosolic acidosis during anoxia. After 80 min of anoxia, pHi of these cells was not different from that of control cells (Table 1). Similarly, no differences were observed when pretreated and control cells from groups 2 and 3 were selectively compared. Reoxygenation during 20 min in medium with pH 7.4 led to recovery of pHi to the initial level. Pretreated cells in group 3 recovered pHi during the initial phase of reoxygenation (first 5 min) significantly faster than control cells. When averaged data from all cells are compared, recovery of pHi was similar for pretreated and control cells.


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  		Table 1 pHi in normoxic, anoxic, and reoxygenated cardiomyocytesa

 
3.5 Effects of treatment with 1,3DOG and BIM
To strengthen the argument that beneficial effects of pretreatment with 1,2DOG are indeed PKC dependent, three additional sets of experiments were designed (Table 2). In the first, treatment of cells with 1,2DOG was substituted by treatment with 20 µmol/l 1,3DOG which is a PKC-inactive isomer of 1,2DOG. In the second, 1 µmol/l BIM, a specific PKC inhibitor, was applied to 1,2DOG-pretreated cells 20 min before and during anoxia until just before reoxygenation. In the third, control cells were similarly treated with 1 µmol/l BIM. Under normoxic conditions treatment with 1,2DOG, 1,3DOG or BIM did not change significantly any of the investigated parameters in comparison to control cells. During anoxia, pretreatment with 1,2DOG led to significant delay of rigor contracture, resulting in a reduction of the time of rigor under anoxia (Table 2). It also caused a reduction of cytosolic Ca2+ overload and prevented hypercontracture upon reoxygenation. Pretreatment with 1,3DOG was not able to reproduce the effects of 1,2DOG. Inhibition of PKC with BIM completely abolished protective effects of 1,2DOG-pretreatment. BIM had no effect when given alone.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
5 Conclusion
 Acknowledgments
 References
 
4.1 The main findings and model features
The aim of the present study was to analyse the mechanism of protection of cardiomyocytes against reoxygenation-induced hypercontracture by a single pre-stimulation of PKC with 1,2DOG. The main findings are the following: pretreatment of cardiomyocytes with the PKC activator 1,2DOG (1) delayed ATP depletion and (2) increased the rate of Ca2+ accumulation during anoxia; (3) the protection against reoxygenation-induced hypercontracture was dependent on the time of ATP depletion and independent of the anoxic changes in cytosolic Ca2+ and pHi.

The model applied here, using isolated cardiomyocytes to analyse some basic aspects of the ischemia-reperfusion scenario, was characterised in several of our previous studies [6,7,10,21]. They were mainly focused on the mechanisms of acute reoxygenation injury, in particular the causes eliciting reoxygenation-induced hypercontracture. Cytosolic Ca2+ overload and acidosis were found to be two important factors affecting hypercontracture. In the present study, the effect of an pre-anoxic activation of PKC on the mechanisms eliciting hypercontracture was investigated. The membrane permeable diacylglycerol, 1,2DOG, was used to activate PKC in cardiomyocytes before their exposure to anoxia. Activation of PKC with 1,2DOG has been shown to be highly specific [22]. In contrast to phorbol esters, which are slowly degraded, diacylglycerols are known to be present only transiently in membranes [23]. Two approaches were used to confirm the specificity of the treatment with 1,2DOG. First, we combined the 1,2DOG-treatment with the presence of the PKC inhibitor BIM and found that this combination abolished the specific effects of 1,2DOG-treatment. Second, we treated cells with the PKC-inactive 1,3DOG and did not observe any of the effects found with 1,2DOG.

4.2 Effect of 1,2DOG-pretreatment on ATP depletion and cytosolic Ca2+ overload during simulated ischemia
It has been shown previously that in cells deprived of oxidative energy production, the onset of rigor contracture coincides with a rapid fall of the cellular ATP content [24]. The coincidence between ATP loss and rigor contracture has been directly demonstrated in single cardiomyocytes injected with luciferase [18]. Unfortunately, the luciferase method cannot be used under anoxic conditions, since the luciferase catalysed oxidation of D-luciferin requires oxygen. Instead, with use of Mg-fura-2, we monitored the rise of the intracellular Mg2+ concentration as this represents an indirect measure of ATP hydrolysis [19,25]. This is because, inside the cell, a major part of Mg2+ is bound to ATP and a fall in ATP concentration causes the release of this part of Mg2+. The Mg-fura-2 ratio started to rise always prior to the onset of rigor contracture. Rigor contracture developed rapidly when the Mg-fura-2 ratio levelled off indicating virtual completion of ATP hydrolysis. This is in agreement with what had been observed with the luciferase method. This sequence of events remained the same when cells had been pretreated with 1,2DOG. Cytosolic pH and cytosolic Ca2+ level, two factors which can modify the Mg-fura-2 ratio, were comparable in control and treated cells at the moment of rigor contracture. Therefore, under either experimental condition the rigor time can be taken to indicate the establishment of extreme ATP depletion.

Treatment before anoxia with the PKC activator 1,2DOG significantly delayed the onset of rigor contracture of cardiomyocytes. Under these conditions about one third of the cells did not develop rigor contracture even after 80 min of anoxia. This observation indicates a pronounced energy sparing effect of 1,2DOG-pretreatment. No difference was observed in lactate production between control and treated cells. The difference in ATP depletion seems thus due to depression of ATP consumption rather than enhancement of anaerobic energy production in the pretreated cells.

It has been reported before that a significant rise in the cytosolic concentration of Ca2+ in energy depleted cardiomyocytes always starts at or shortly after the time of rigor contracture [7,8]. The same was observed in the present study. The delay in rigor contracture after 1,2DOG-pretreatment was thus associated with a delayed onset of cytosolic Ca2+ accumulation. Once rigor contracture occurred in pretreated cardiomyocytes, however, they accumulated cytosolic Ca2+ significantly faster than control cells. This resulted in an augmented cytosolic Ca2+ overload at the end of 80 min anoxia when cells with similar rigor times were compared (groups 2 and 3). Only in pretreated cells which developed rigor contracture at times later than 60 min of anoxia (group 4), a lower degree of cytosolic Ca2+ overload at the end of 80 min anoxia was observed. This latter finding is likely due to the shortage of time (<20 min) between onset of Ca2+ accumulation (rigor) and reoxygenation. Group 5 contained the cells pretreated with 1,2DOG which did not develop rigor contracture at all and, therefore, did not start to accumulate Ca2+ within 80 min anoxia. When grouping for the highly variable rigor times is neglected and all data are taken together to obtain the average Ca2+ overload during 80 min anoxia (Table 2), 1,2DOG-pretreatment appears to reduce anoxic Ca2+ overload.

It was found previously [26], that the cause of Ca2+ accumulation in this model is the activation of the reverse mode of the Na+/Ca2+ exchanger. The activity of this exchanger, both in forward and reverse modes, is under phosphorylation control of PKC [27]. Therefore, it might be speculated that the acceleration of Ca2+ accumulation after ATP depletion in cells pretreated with PKC activator is due to enhanced phosphorylation of the sarcolemmal Na+/Ca2+ exchanger.

A short methodological consideration seems required at this point. We indirectly estimated the changes in cytosolic Ca2+ concentration by determination of the fura-2 ratio. The fura-2 ratio may be influenced by differences in pHi [28]. This cannot account for the differences in the fura-2 ratio at the end of anoxia, however, since at this point pHi was the same in control and pretreated cells (Table 1).

4.3 Effect of 1,2DOG-pretreatment on reoxygenation-induced hypercontracture
Pretreatment with 1,2DOG before anoxia significantly reduced reoxygenation-induced hypercontracture (Table 2), but the degree of protection was dependent on the time of rigor contracture. In pretreated cardiomyocytes which developed rigor contracture early during anoxia, i.e. cells of group 2, hypercontracture was similar to that in control cells. Hypercontracture was, however, significantly reduced in cells which developed rigor contracture after 40 min of anoxia or later (groups 3–5)

The most interesting finding of this study is the reduction of hypercontracture in pretreated cells of group 3. It has been found in previous studies that Ca2+ overload is an important causal factor for reoxygenation-induced hypercontracture [9]. The data of the present study also exhibit a correlation between the end-anoxic fura-2 ratio and hypercontracture in control cells. In contrast, no such correlation was observed in pretreated cells. These data indicate that PKC stimulation activates some mechanism which provides protection against hypercontracture even at a high cytosolic Ca2+ level. It has been shown previously that prolonged acidosis during the early phase of reoxygenation can protect against hypercontracture [7,20]. This cannot account for the protection of pretreated, Ca2+ overloaded cells of group 3, however, because the recovery of pHi during reoxygenation proceeded even faster in pretreated than in control cells of the same group. One may speculate that protection against reoxygenation-induced hypercontracture after activation of PKC is due to attenuation of the dephosphorylation of some proteins during anoxia [21], but the present study was not designed to test this specific hypothesis.

4.4 Comparison with preconditioning in the whole heart
Although the results of the present study cannot be directly extrapolated to the phenomenon of ischemic preconditioning in the whole heart, some effects of ischemic preconditioning can be compared with the effects of 1,2DOG-pretreatment on isolated cardiomyocytes. It has been demonstrated in numerous studies that a decrease in the rate of ATP depletion takes part in the protective mechanisms of ischemic preconditioning [29,30]. This is in agreement with the attenuation of anoxic ATP depletion in pretreated cardiomyocytes observed in the present study. Another beneficial effect of ischemic preconditioning in the whole heart consists in a reduction of Ca2+ overload during sustained ischemia [31]. A reduction of anoxic Ca2+ overload was also observed in the present study when the data of all cells were averaged (Table 2). The detailed temporal analysis of Ca2+ accumulation among the individual cells has revealed, however, that the impression that 1,2DOG-pretreatment directly attenuates anoxic Ca2+ overload is misleading.

Ischemic preconditioning represents an effective means to reduce the extent of post-ischemic tissue necrosis [32]. Post-ischemic necrosis may in part be due to reperfusion-induced injury of which reoxygenation-induced hypercontracture is a major contributing cause [12,33,34]. The question has not yet been addressed whether ischemic preconditioning of myocardium exerts part of its protective action through a specific effect on reperfusion-induced injury. Results of the present and a previous study [6] show that PKC preactivation protects myocardial cells specifically against this important mechanism of reoxygenation-induced injury.


   5 Conclusion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusion
Acknowledgments
 References
 
The results of the present study show that a single preactivation of PKC in isolated adult cardiomyocytes leads to beneficial (delay of ATP depletion) as well as to detrimental (acceleration of cytosolic Ca2+ overload) effects during anoxia. Because of the dominance of the energy-sparing effect, the overall result of both effects is favourable (reduction of an average anoxic Ca2+ overload). Reoxygenation-induced hypercontracture is significantly reduced after PKC preactivation. This protection during reoxygenation is independent of changes in cytosolic Ca2+ and pHi.

Time for primary review 28 days.


   Acknowledgments
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusion
 Acknowledgments
References
 
The technical help of D. Schreiber and H. Holzträger is gratefully acknowledged. This work was support by the BIOMED-2 program of the European Union and Grant LA 1159/2-1 of the Deutsche Forschungsgemeinschaft.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusion
 Acknowledgments
 References
 

  1. Liu Y., Ytrehus K., Downey J.M. Evidence that translocation of protein kinase C is a key event during ischemic preconditioning of rabbit myocardium. J Mol Cell Cardiol (1994) 26:661–668.[CrossRef][ISI][Medline]
  2. Speechly Dick M.E., Mocanu M.M., Yellon D.M. Protein kinase C. Its role in ischemic preconditioning in the rat. Circ Res (1994) 75:586–590.[Abstract/Free Full Text]
  3. Mitchell M.B., Meng X., Ao L., Brown J.M., Harken A.H., Banerjee A. Preconditioning of isolated rat heart is mediated by protein kinase C. Circ Res (1995) 76:73–81.[Abstract/Free Full Text]
  4. Chen W., Wetsel W., Steenbergen C., Murphy E. Effect of ischemic preconditioning and PKC activation on acidification during ischemia in rat heart. J Mol Cell Cardiol (1996) 28:871–880.[CrossRef][ISI][Medline]
  5. Armstrong S., Ganote C.E. Preconditioning of isolated rabbit cardiomyocytes: effects of glycolytic blockade, phorbol esters, and ischaemia. Cardiovasc Res (1994) 28:1700–1706.[Abstract/Free Full Text]
  6. Ladilov Y.V., Balser C., Piper H.M. Protection of rat cardiomyocytes against simulated ischemia and reoxygenation by treatment with protein kinase C activator. Circ Res (1998) 82:451–457.[Abstract/Free Full Text]
  7. Ladilov Y.V., Siegmund B., Piper H.M. Protection of reoxygenated cardiomyocytes against hypercontracture by inhibition of Na+/H+ exchange. Am J Physiol (1995) 268:H1531–H1539.[ISI][Medline]
  8. Piper H.M., Siegmund B., Ladilov Y.V., Schluter K.D. Calcium and sodium control in hypoxic-reoxygenated cardiomyocytes. Basic Res Cardiol (1993) 88:471–482.[CrossRef][ISI][Medline]
  9. Miyata H., Lakatta E.G., Stern M.D., Silverman H.S. Relation of mitochondrial and cytosolic free calcium to cardiac myocyte recovery after exposure to anoxia. Circ Res (1992) 71:605–613.[Abstract/Free Full Text]
  10. Piper H.M., Siegmund B., Schlüter K.D. Prevention of the oxygen paradox in the isolated cardiomyocyte and the whole heart. Am J Cardiovasc Pathol (1992) 4:115–122.[Medline]
  11. Vander Heide R.S., Angelo J.P., Altschuld R.A., Ganote C.E. Energy dependence of contraction band formation in perfused hearts and isolated adult myocytes. Am J Pathol (1986) 125:55–68.[Abstract]
  12. Ganote C.E. Contraction band necrosis and irreversible myocardial injury. J Mol Cell Cardiol (1983) 15:67–73.[ISI][Medline]
  13. Piper H.M., Probst I., Schwartz P., Hütter F.J., Spieckermann P.G. Culturing of calcium stable adult cardiac myocytes. J Mol Cell Cardiol (1982) 14:397–412.[CrossRef][ISI][Medline]
  14. Koop A., Piper H.M. Protection of energy status of hypoxic cardiomyocytes by mild acidosis. J Mol Cell Cardiol (1992) 24:55–65.[ISI][Medline]
  15. Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem (1976) 72:248–254.[CrossRef][ISI][Medline]
  16. Allshire A., Piper H.M., Cuthbertson K.S., Cobbold P.H. Cytosolic free Ca2+ in single rat heart cells during anoxia and reoxygenation. Biochem J (1987) 244:381–385.[ISI][Medline]
  17. Wallenstein S., Zucker C.L., Fleiss J.L. Some statistical methods useful in circulation research. Circ Res (1980) 47:1–9.[Abstract/Free Full Text]
  18. Bowers K.C., Allshire A.P., Cobbold P.H. Bioluminescent measurement in single cardiomyocytes of sudden cytosolic ATP depletion coincident with rigor. J Mol Cell Cardiol (1992) 24:213–218.[ISI][Medline]
  19. Silverman H.S., Di Lisa F., Hui R.C., et al. Regulation of intracellular free Mg2+ and contraction in single adult mammalian cardiac myocytes. Am J Physiol (1994) 266:C222–C233.[ISI][Medline]
  20. Piper H.M., Balser C., Ladilov Y.V., et al. The role of Na+/H+ exchange in ischemia-reperfusion. Basic Res Cardiol (1996) 9:191–202.
  21. Ladilov Y.V., Siegmund B., Balser C., Piper H.M. Simulated ischemia increases the susceptibility of rat cardiomyocytes to hypercontracture. Circ Res (1997) 80:69–75.[Abstract/Free Full Text]
  22. Capogrossi M.C., Kaku T., Filburn C.R., et al. Phorbol ester and dioctanoylglycerol stimulate membrane association of protein kinase C and have a negative inotropic effect mediated by changes in cytosolic Ca2+ in adult rat cardiac myocytes. Circ Res (1990) 66:1143–1155.[Abstract/Free Full Text]
  23. Hee Cheong M., Severson D.L. Metabolism of dioctanoylglycerol by isolated cardiac myocytes. J Mol Cell Cardiol (1989) 21:829–837.[CrossRef][ISI][Medline]
  24. Haworth R.A., Hunter D.R., Berkoff H.A. Contracture in isolated adult rat heart cells. Role of Ca2+, ATP, and compartmentation. Circ Res (1981) 49:1119–1128.[Abstract/Free Full Text]
  25. Leyssens A., Nowicky A.V., Patterson L., Crompton M., Duchen M.R. The relationship between mitochondrial state, ATP hydrolysis, [Mg2+]i and [Ca2+]i studied in isolated rat cardiomyocytes. J Physiol Lond (1996) 496:111–128.[Abstract/Free Full Text]
  26. Haffner S, Ladilov Y, Balser C, Piper HM. Inhibition of the reverse mode of the Na+/Ca2+ exchange protects cardiomyocytes against anoxia-reoxygenation injury (Abstract). Pflügers Arch 1998;435 Suppl:R200.
  27. Iwamoto T., Pan Y., Wakabayashi S., et al. Phosphorylation-dependent regulation of cardiac Na+/Ca2+ exchanger via protein kinase C. J Biol Chem (1996) 271:13609–13615.[Abstract/Free Full Text]
  28. Harrison S.M., Frampton J.E., McCall E., Boyett M.R., Orchard C.H. Contraction and intracellular Ca2+, Na+, and H+ during acidosis in rat ventricular myocytes. Am J Physiol (1992) 262:C348–C357.[ISI][Medline]
  29. Murry C.E., Richard V.J., Reimer K.A., Jennings R.B. Ischemic preconditioning slows energy metabolism and delays ultrastructural damage during a sustained ischemic episode. Circ Res (1990) 66:913–931.[Abstract/Free Full Text]
  30. Kida M., Fujiwara H., Ishida M., et al. Ischemic preconditioning preserves creatine phosphate and intracellular pH. Circulation (1991) 84:2495–2503.[Abstract/Free Full Text]
  31. Steenbergen C., Perlman M.E., London R.E., Murphy E. Mechanism of preconditioning. Ionic alterations. Circ Res (1993) 72:112–125.[Abstract/Free Full Text]
  32. Li Y.W., Whittaker P., Kloner R.A. The transient nature of the effect of ischemic preconditioning on myocardial infarct size and ventricular arrhythmia. Am Heart J (1992) 123:346–353.[CrossRef][ISI][Medline]
  33. Schlack W., Uebing A., Schafer M., et al. Regional contractile blockade at the onset of reperfusion reduces infarct size in the dog heart. Pflugers Arch (1994) 428:134–141.[CrossRef][ISI][Medline]
  34. Garcia-Dorado D., Theroux P., Duran J.M., et al. Selective inhibition of the contractile apparatus. A new approach to modification of infarct size, infarct composition, and infarct geometry during coronary artery occlusion and reperfusion. Circulation (1992) 85:1160–1174.[Abstract/Free Full Text]

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Adenine nucleotide translocator, a mitochondrial carrier protein, and fate of cardiomyocytes after ischaemia/reperfusion
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