© 1997 by European Society of Cardiology
Copyright © 1997, European Society of Cardiology
Rigor tension in single skinned rat cardiac cell: role of myofibrillar creatine kinase
Laboratoire de Cardiologie Cellulaire et Moléculaire INSERM U-446, Faculté de Pharmacie, Université Paris-Sud, Châtenay-Malabry 92296, France
* Corresponding author. Tel. (+33) 146835763; Fax (+33) 146835475.
Received 31 December 1996; accepted 26 June 1997
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Abstract |
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Objective: To elucidate the role of bound creatine kinase in adenine nucleotide compartmentation in myofibrils, the effects of this enzyme's substrates and products on rigor tension were studied in using isolated skinned rat cardiomyocytes rather than fibres, to avoid restrictions due to concentration gradients within the multicellular preparations. Methods: A new experimental set-up was built to allow continuous and stable measurements of force developed by cells. Triton X-100-treated cardiomyocytes were glued between a glass holder and the needle of a galvanometer. A feedback system allowed the precise measurement of force by recording the coil current necessary to prevent movement of the needle. Results: At very low [Ca2+] (pCa 7), as MgATP level decreased, rigor tension appeared. In the absence of phosphocreatine (PCr), this tension started to rise at MgATP concentrations several times higher than in the presence of 12 mM PCr. In the absence of PCr, the pMgATP/tension curves of single cells usually had a complicated relationship which could not be analyzed by a simple Hill equation. In the absence of PCr, 250 µM MgADP strongly potentiated rigor tension development in the 1 mM–3 µM range of [MgATP]; at 100 µM MgATP, in the presence of MgADP, the tension was 4.6 times higher than in the absence of MgADP. Addition of 12 mM PCr immediately eliminated rigor. Finally, in the presence of 100 µM MgATP and 250 µM MgADP, a decrease in PCr resulted in rigor; the half-maximal contracture being recorded at 1 mM PCr. Conclusions: These results indicate a myofibrillar compartmentation of adenine nucleotides influenced by bound creatine kinase, since at equal MgATP concentrations in extramyofibrillar milieu the response of myofibrils strongly depends on the presence of PCr. Local accumulation of ADP in myofibrils due to a fall in cellular PCr and inability of myofibrillar creatine kinase to rephosphorylate ADP produced by myosin ATPase could be an important mechanism of diastolic tension rise in ischaemic conditions.
KEYWORDS Creatine kinase; Phosphocreatine; Adenine nucleotides; Intracellular compartmentation; Ischaemic contracture; Rat ventricular cells
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1 Introduction |
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TopAbstract1 Introduction2 Materials and methods3 Results4 DiscussionReferences |
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Contraction of muscle results from the cyclic interaction between myosin and actin. This interaction is driven by the concomitant hydrolysis of ATP (in the form of MgATP) by myosin. The mechano-chemical transduction includes several biochemical steps: the binding of MgATP to actomyosin, the cleavage of ATP, the release of inorganic phosphate and subsequent release of ADP.
A decrease in MgATP concentration in myofibrils is known to induce myosin crossbridge formation which, in turn, results in force generation. This force, which develops in the absence of Ca2+, is called rigor tension. Alterations in the energy supply of myofibrils are also able to considerably change parameters of Ca-activated tension [1, 2]. It is thus clear that providing the myosin ATPase with energy has critical importance for efficient muscle functioning.
Numerous studies have suggested a very important role for myofibrillar creatine kinase (CK) in myosin ATPase energy supply (for review, see [3]). CK catalyses the reversible transfer of a phosphate moiety between phosphocreatine (PCr) and MgADP. A hypothesis has been put forward that MgATP produced by bound CK has preferential access to the myosin ATPase (‘functional coupling’) as compared to MgATP by diffusing from the extramyofibrillar space (for review, see [3, 4]). As a consequence, under conditions of PCr deficiency, the inability of the myofibrillar CK to locally rephosphorylate ADP (for example in ischaemia) may lead to a local drop in ATP and an accumulation of ADP in myofibrils. These changes may be a crucial factor determining the appearance of myocardial contracture under conditions of energy deficiency when PCr reserves are depleted but cellular ATP content is still rather high.
To study the role of myofibrillar CK in the regulation of cardiac muscle mechanical activity, it is very important to achieve strict control of high energy phosphate concentrations in the extramyofibrillar milieu. Such a control can not be adequately performed using conventional preparations of skinned fibres due to the problem of intercellular diffusion. It is probable that diffusion limitations (due to the size of the preparations) and marked ATP consumption inside skinned fibres even in the virtual absence of Ca2+ [5]would produce considerable ATP and ADP gradients between the bulk solution and the core of preparations. In other words, the resultant intercellular gradients could contribute more than intracellular compartmentation of the adenine nucleotides to rigor tension development. Therefore, the only approach that allows us to control the intracellular medium and to keep the cellular architecture intact is to use single skinned cells.
The aim of the present study was to investigate the role of bound CK in the regulation of rigor tension developed by single skinned cardiac cells. The data obtained show that even in single cells, CK reaction metabolites —PCr and ADP— have a profound influence on the ATP/rigor tension dependence, thus indicating a myofibrillar compartmentation of adenine nucleotides controlled by bound CK. Accumulation of ADP in the myofibrillar compartment under conditions of PCr deficiency may be the basis for myocardial ischaemic contracture.
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2 Materials and methods |
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2.1 Preparation of cardiac myocytes
All experiments reported here conform 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). Single myocytes were isolated using collagenase digestion of the adult rat heart, as described earlier [6]. Male Wistar rats (225–250 g) were anaesthetized with ethyl carbamate 20% (1 ml/100 g body wt). The hearts were removed and the coronary arteries perfused with oxygenated Krebs solution containing (mM): NaCl, 117; KCl, 5.7; NaHCO3, 4.4; KH2PO4, 1.5; MgCl2, 1.7; HEPES, 21; glucose, 11; creatine, 10; taurine, 20; insulin, 2.1 IU/l (pH 7.0, 37°C) at 6 ml/min for 5 min with no added Ca2+ and at 4 ml/min for 60 min with 20 µM Ca2+ and 1–1.2 mg/ml collagenase A (0.32 U/mg). The atria were then removed and the ventricles were cut into small pieces that were gently dissociated with forceps in the same medium without collagenase. The cells were then filtered and incubated for 15 minutes. After decanting the supernatant, the myocytes were resuspended in the Krebs solution (pH 7.4 at 37°C) with 1 mM Ca2+ and 0.5% bovine serum albumin. Before skinning 50–80% of the cells isolated with this procedure were calcium tolerant, whereas after skinning, cells responded to calcium with a vigorous contraction.
Cells were skinned in the relaxing solution (see below) with 0.3% (v/v) Triton X-100 for 5 min in Eppendorf tubes. The myocytes were then centrifuged and placed in glass Petri dishes containing relaxing solution (3 ml).
Data presented in this work were obtained on 47 single ventricular cells. Mean cell width and length were 16±1 and 94±3 µm, respectively.
2.2 Mechanics
The mechanical set-up was built around an inverted microscope (Olympus, Tokyo) that was placed on top of a vibration isolation table (Micro-G, Technical Manufacturing). Cells were attached at one end to the needle of a galvanometer. A flag glued to the needle had a slit through which a light beam from a lamp passed to a light detector. A feedback system used the light-induced current to keep the position of the galvanometer needle constant. Recording the coil current necessary to prevent the needle movements allowed us to measure force developed by cells under conditions very close to isometric ones (maximal Ca-activated tension resulted in cell shortening not more than few percent of the cell length). The time resolution of the force detection system was about 10 ms. By use of a video camera (model AVC-D5CE, Sony, Japan), cell width and length were monitored.
For cell attachment to the set-up, we used the tip of a minutien pin and glass micropipettes. The pin was glued to the end of the galvanometer needle. The glass micropipette with the stainless steel holder was mounted onto a micromanipulator (Narishige, Japan) to allow precise three-way positioning. Single cardiomyocytes were attached to the pin at one end and to the tip of a micropipette at the other, with adhesive ‘Great Stuff’ (Insta-Foam Products), as proposed by Sweitzer and Moss [7]. The tip of the pin and that of the micropipette were coated with the foam diluted in acetone, and gently placed on the ends of the cardiomyocyte in solution. Before stretching the cell, ample time (>45 min) was allowed for the glue to set.
Rigor tension was shown to be dependent on the order of metabolic interventions. The relative tension at any intermediate [ATP] could vary depending on whether myofilaments were initially in a relaxed or rigor state [8]. Evidently then, existing rigor crossbridges could influence the formation of new ones. During the determination of pMgATP/tension relationships, it therefore seemed important to avoid mechanical perturbations during solution changes. Such perturbations could break the crossbridges and thus affect rigor development in an unpredictable manner. We used a system for cell perfusion that eliminated force transients due to the surface tension effect during the passage of the cell from one solution to another; in this way we were able to obtain stable force baselines.
Cell perfusion with various solutions was carried out by placing the cells at the opening of a 250-µm inner diameter capillary from which the solutions were flowing at a rate of 80–90 µl/min. The position of the capillary was adjusted with a micromanipulator. The solutions flowed through thin Teflon tubes connected to a series of 8 or 16 syringes. This system thus allowed us to perfuse the same cell with up to 16 solutions of different compositions. To overcome the tube's resistance and increase the flow, a pressure of about 0.4 atm was applied to the syringes. Opening and closing clamps on the tubes allowed us to change perfusion solutions rapidly (less than 2–3 s) and completely. An aspiration tube was used to evacuate extra fluid from the Petri dish and thus to keep the fluid level constant. All the experiments were performed at 20–22°C.
2.3 Solutions and reagents
Solutions (Table 1) were calculated by use of the computer program of Fabiato [9]to contain (mM): EGTA, 10; imidazole, 30; Na+, 30; free Mg2+, 1; dithiothreitol, 0.3; ionic strength was adjusted to 0.16 M with potassium acetate. In all solutions pH (7.1) was adjusted with acetic acid. To keep [Ca2+] close to the diastolic intracellular value, in rigor solutions, pCa (the negative logarithm of free Ca2+ concentration) was 7. In relaxing and activating solutions, pCa's were 9 and 4.5 respectively. Relaxing and activating solutions also contained 3.16 mM MgATP (pMgATP, negative logarithm of [MgATP], 2.5) and 12 mM PCr. Rigor solutions were obtained by mixing two solutions with (3.16 mM) and without MgATP, in the presence or absence of 12 mM PCr or 250 µM MgADP.
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Collagenase A was purchased from Boehringer, PCr (Neoton, Sciapparelli Farmaceutica, Turin, Italy) was a kind gift of Prof. E. Strumia. Other reagents were obtained from Sigma Chemical, St. Louis, Mo.
2.4 Validation of the single cell technique
To test the ability of our set-up to ensure adequate cell perfusion and isometric force determination and to characterise mechanical activity of skinned cells, parameters of Ca-activated tension were initially estimated. Fig. 1 shows isometric force traces for a single myocyte at different pCa values. After reaching the maximal tension at pCa 4.5 (usually 6–8 µN), perfusion with the relaxing solution (pCa 9) induced rapid relaxation of the cell. Stepwise increase in Ca2+ concentration in perfusing solutions led to stepwise tension development. Two consequent applications of the solutions with increasing [Ca2+] gave almost the same results thus indicating the reproducibility of the experiments.
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pCa/tension relationships in single cells were analyzed by using the Hill equation. Cells stretched by 12% over the slack length (sarcomere length 2.05–2.10 µm) showed a pCa50 (pCa for half-maximal activation) and Hill coefficient to be 5.564±0.048 and 2.74±0.14 respectively (n = 16). Cells responded to stretching by a slight but significant increase in calcium sensitivity. After stretching from 5 to 12% over the slack length, pCa50 rose by 0.060±0.016 (p<0.02; n = 5). Further increase of cell length up to 20% over the slack length (sarcomere length 2.15–2.25 µm) led to some additional rise in calcium sensitivity of borderline significance (by 0.044±0.025; p = 0.055; n = 4). Thus, parameters of mechanical activity and responses to stretching obtained in our experiments on single skinned cardiomyocytes were very close to those reported by other authors [10, 11].
These data showed that our cell perfusion system created a sufficient volume of test solution around the cells so that there was no contact with the bulk solution. The normal pCa/tension curves also demonstrated that there was no loss of troponin C from our preparations of skinned cells. This could have been important since it has been shown [8]that even partial extraction of troponin C leads to marked changes in [MgATP]/rigor tension relationship.
2.5 Experimental protocol
Rigor tension development was studied after stretching cells by 12% over their slack length. Initially, each cell was challenged by several consequent applications of the activating (pCa 4.5) and relaxing solutions. This was done for two reasons. Firstly, rapid force development (after activation) and rapid relaxation (after starting perfusion with the relaxing solution) meant that the position of the perfusing capillary was correct, and the cell was situated completely in the perfusion solution. Secondly, the value of maximal Ca-activated force obtained just before rigor tension measurement was used for normalization of the rigor force. Such an approach for normalization was chosen because very often pMgATP/tension relationships had complicated forms and could not be analyzed with a simple Hill equation. Furthermore, the [MgATP] at which maximal rigor tension was developed, varied between experiments. Maximal Ca-activated force was determined before each series of rigor determinations, and if in the course of the experiment the mechanical activity had a tendency to decay, the maximal Ca-activated tension measured just before this series was taken for rigor force normalization. Usually, a control activation with pCa 4.5 was performed at the end of the rigor protocol, and if the maximal force fell by more than 20%, the data were rejected.
Active and rigor tension values were taken as the difference between the total active or rigor tension, and the tension in the relaxing solution.
2.6 Statistical analysis
Values were expressed as mean±SEM. Data concerning effects of cell stretching on calcium sensitivity were tested by paired t-test. Threshold values for rigor tension development were compared with a one-way ANOVA followed by Dunn t-test. Effects of PCr and ADP on pMgATP/tension curve as well as effect of diadenosine pentaphosphate on pMgADP/tension curve were evaluated by use of a repeated measures two-way ANOVA. Statistical significance was considered to be reached at P<0.05.
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3 Results |
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3.1 Effects of MgATP and PCr on rigor tension development
Following activation at pCa 4.5 (Fig. 2), cells developed maximal tension and then relaxed at pCa 9. To establish a pMgATP/tension relationship, [MgATP] was stepwise decreased from 3.16 mM (pMgATP 2.5) to 3.16 µM (pMgATP 5.5). [Ca2+] was set to be as low as 100 nM, much lower than the threshold for active tension development. In the absence of PCr, very often a weak rigor force appeared often at a [MgATP] in the range of 1 mM–0.1 mM (pMgATP 3–4). At [MgATP] below 0.1 mM a much higher rigor tension was observed. Fig. 2A shows that the rise in rigor force with a decrease in MgATP was often not monotonic. In this example, the tension developed at pMgATP 3.75 was considerably higher than at pMgATP 4. Such deviations from the expected Hill relationship were observed in many cells perfused with the solutions not containing PCr. Sometimes application of a solution with lower [MgATP] induced fluctuations in force before a definite level was established. Usually, such unstable rigor force was observed at pMgATP lower that 4.5–4.75. Control experiments showed that simple switching between syringes containing the same pMgATP level did not provoke marked mechanical artefacts (not shown).
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Addition of 12 mM PCr to the perfusion solutions essentially changed the pMgATP/tension relationship. Fig. 2B representing a trace of rigor force of a single cell shows that the tension appeared only at very low [MgATP]. The stepwise decrease in [MgATP] always induced a monotonic enhancement of rigor. The mean threshold values for rigor tension (pMgATP at which the tension exceeded 10% of the maximal force) were significantly different in the presence and absence of PCr (4.91±0.10, n = 12 and 3.75±0.18, n = 17; p<0.01).
Fig. 3A demonstrates the influence of bound CK/PCr system on rigor tension development. Decrease of [MgATP] to 18 µM (pMgATP 4.75) in the absence of PCr resulted in a relatively high rigor force, about 50% of the maximal Ca-activated force. At the same [MgATP], PCr addition immediately induced relaxation of the cell, so that rigor force became very low. Withdrawal of PCr led to restoration of the rigor tension.
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Effect of a stepwise decrease in [PCr] in the presence of 100 µM MgATP and 250 µM MgADP is shown in Fig. 3B. No tension was observed at [PCr] as low as 5 mM. A further decrease in [PCr] resulted in a steep rise of rigor tension. Half-maximal force appeared at a [PCr] close to 1 mM. Complete withdrawal of PCr was followed by a development of rigor force, being much higher than tension at 100 µM MgATP in the absence of exogenous ADP (Fig. 2A). These results imply that the presence of ADP could play an important role in rigor tension development when PCr reserves are exhausted.
3.2 Effect of ADP on rigor tension development
The depletion of PCr results in an inability of myofibrillar CK to rephosphorylate ADP generated in the ATPase reaction and thus to an accumulation of this compound. Therefore, one may suggest that early appearance of rigor in the absence of PCr could be related not only to the decrease in [ATP] in myofibrils but also to the local increase in [ADP]. To study if ADP could modulate pMgATP/tension relationship in a single cell, we investigated the influence of exogenously added 250 µM MgADP on rigor tension.
In the presence of 12 mM PCr and ADP, no rigor tension could be elicited even in the complete absence of MgATP in the perfusion solutions. This shows the effective rephosphorylation capacity of myofibrillar CK. However, in the absence of PCr, ADP significantly potentiated rigor tension. Fig. 4A shows that the threshold for force appearance was shifted to lower values of [MgATP]. A marked rigor was observed at [MgATP] as high as 0.56 mM (pMgATP 3.25). At pMgATP 4.5, the rigor tension developed by the cell reached approximately the same amplitude as the maximal Ca-activated force. The mean threshold pMgATP values for rigor tension were significantly different in the presence and absence of ADP (3.09±0.09, n = 19 and 3.75±0.18, n = 17; p<0.05)
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Effect of ADP addition at the same [MgATP] is shown in Fig. 4B. Decrease of [MgATP] to 100 µM in the absence of PCr led to the appearance of relatively low rigor tension (about 15% of maximal Ca-activated tension). However, addition of 250 µM MgADP induced a considerable rise in rigor force up to 75% of the maximal tension. Withdrawal of ADP immediately reversed its effect.
Fig. 5 shows averaged values of relative rigor tension as a function of pMgATP, and effects of PCr and ADP. One can see that in the absence of PCr, a small force appeared at relatively low pMgATP, which rose only slightly as pMgATP increased up to 4–4.25. However, at pMgATP values above 4, higher rigor tension started to develop. MgADP (250 µM) potentiated the rigor force considerably. Even at pMgATP as low as 3, ADP markedly enhanced rigor tension.
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Conversely, the presence of 12 mM PCr very significantly diminished the rigor force in the range of pMgATP 4–5.25. Marked rigor tension appeared only at pMgATP 4.75–5. It is interesting to note that in the presence of PCr, when ADP is eliminated from the intracellular space, variations in the individual values are much smaller.
The role of ADP in potentiating rigor tension was also studied in experiments where [MgADP] was stepwise increased in the presence of a relatively high concentration of MgATP (1 mM). The results are presented in Fig. 6. It can be seen that the increase in [MgADP] resulted in an almost linear rise in rigor force. In some experiments, diadenosine pentaphosphate (100 µM), an inhibitor of myokinase, was added to check if this enzyme bound to myofibrils played a role in decreasing local [ADP]. Indeed, the inhibition of myokinase resulted in higher rigor tension, thus indicating that endogenous myokinase could participate in increasing the local ATP/ADP ratio in myofibrils.
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4 Discussion |
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This work represents the first study of the role of myofibrillar CK in regulation of the cardiac cells mechanical activity with a strict control of the intracellular medium. The data obtained show that (1) as [MgATP] decreases, the rigor tension development depends on the presence of PCr, and (2) absence of PCr allows ADP to essentially affect the force developed by cells.
In this work we studied the effects of decrease in high energy phosphates on the tension developed by skinned cells. As far as the experiments have been performed at pCa 7, and the decrease in the ATP/ADP ratio augments the Ca2+ sensitivity of myofibrils, one can not exclude that active, Ca2+-stimulated crossbridges could contribute to the rise in force. However, data obtained in skinned fibres in the absence of Ca2+ [12, 13]suggest that ADP is able to considerably modulate ‘pure’ rigor tension.
4.1 Effect of PCr on pMgATP/tension relationship
One of the main results of this study is the influence of PCr on rigor tension developed, evidently due to activity of CK bound to myofibrils. In the presence of 12 mM PCr, rigor tension only developed at very low MgATP concentrations. Similar results have been shown for rigor tension developed by skinned rat ventricular fibres having diameters an order of magnitude higher [12, 14]. This is in good agreement with apparent Km values for MgATP obtained for the cardiac myosin ATPase of isolated myofibrils in the presence of PCr [15]. Evidently, myofibrillar CK in the presence of 12 mM PCr can remove any diffusion barrier for MgATP between the vicinity of myosin ATPase and the medium around preparations, whatever the diameter of the latter.
In the absence of PCr, however, rigor tension development showed completely different characteristics. Qualitatively similar to what has been demonstrated in skinned fibres [12, 14, 16, 17], force appeared for smaller decreases in MgATP concentrations higher than in the presence of PCr. These results clearly showed that the effects of PCr on rigor tension developed by skinned fibres could not be simply explained by removal of ATP/ADP concentration gradients between the cellular layers in multicellular preparations. Evidently, even inside a single cell, a compartmentation of adenine nucleotides exists in myofibrils, which is controlled by bound CK.
Previous data on the influence of the PCr/CK system on rigor tension developed by single cardiac cells have been sparse. Thus, Fabiato and Fabiato [1]investigated the pMgATP/tension relationship in single cells in the absence of PCr, and to buffer intracellular [MgATP] the authors used a relatively high total [ATP] whilst varying total Mg concentration. Despite such a buffering, they found that at pMgATP 4.5 the cells developed a marked tension; however, in the presence of PCr even multicellular preparations showed no significant rigor at this pMgATP value. Experiments made on a suspension of digitonin-permeabilised cardiomyocytes [18]also demonstrated that in absence of PCr, the energy-deprived contracture developed at a relatively high [MgATP] (>100 µM).
Recently, the pMgATP/tension relationship was investigated in single cells in the absence of PCr [8]. The author found that rigor force appeared at pMgATP>4.5 and claimed that "in two control experiments, PCr (14.5 mM) and PCr+creatine phosphokinase (150 units/ml) were added to the solutions and the results obtained were found to be comparable with those without added PCr or CPK". Since these results were not shown in the paper, it is difficult to estimate possible differences observed. Furthermore, in this study, to obtain the tension baseline, releases and re-extensions of myocyte length were performed after changing solutions; such perturbations could influence rigor tension and may restrict the measurement of a small rigor force. Also, this study was carried out at 15°C, using nominally Ca2+-free medium, whereas we worked at 22°C and used a more physiological [Ca2+] (100 nM) which could result in a higher ATPase activity and a consequential facilitation of adenine nucleotide compartmentation.
In contrast to the study of Metzger [8], the importance of the CK reaction in preventing rigor in cells has been demonstrated by Nichols and Lederer [19]. They found that saponin-permeabilised cardiomyocytes at pCa 7 had a [MgATP] threshold for rigor contracture of 1 mM in the absence of PCr, but only 100 µM in the presence of PCr. Maximal speed of contracture development strongly depended on PCr, being much higher in its absence. These data thus suggest the existence of an intracellular gradient of adenine nucleotides which could be overcome by CK functioning.
4.2 Rigor tension in absence of PCr and possible role of local ADP accumulation
We found that in the absence of PCr, individual pMgATP/tension relationships in cells often demonstrated a non-monotonic behaviour. A similar observation was made by Nichols and Lederer (Fig. 4 in [19]) who found that increasing pMgATP from 3 to 3.75 induced a rise in the maximal speed of cellular contracture development, but further pMgATP increase to 4 did not augment this parameter at all, which, however, did rise dramatically again at pMgATP>4.25. The tension at higher [MgATP]s probably depends more on local [ADP] at the myofibrils. Indeed, our results show that ADP has a very strong effect on rigor tension. It is therefore very likely that rigor force in the absence of PCr, at relatively high [MgATP] (that is substrate for actomyosin ATPase), could be determined mainly by a local accumulation of ADP in the myofibrillar compartment (due to a high ATPase activity) rather than by a simple decrease in local [MgATP]. The latter event seems to contribute more to rigor force at low (<100 µM) [MgATP]. This might explain the appearance of two different parts of the pMgATP/tension curve and the non-monotonic rise in force.
4.3 Local [MgATP] and [MgADP] in cardiac myofibrils and ischaemic contracture
Mechanisms of development of ischaemic contracture, i.e. gradual rise in diastolic stiffness and force, have been extensively discussed in the literature ([14, 20, 21]and references therein) but the fundamental basis of this event is still not completely clear. Numerous data indicated the important role of rigor crossbridge formation. These results, however, were in apparent contradiction with the well-known fact that total ATP content in myocardium is relatively high at the moment of the ischaemic contracture development (10–50% of the preischaemic values according to NMR-based estimates [21, 22]). To explain this, it was proposed that ATP is compartmentalised, and [MgATP] in myofibrils differs from that in the cytosol.
The results of the present study confirm this latter hypothesis but, in addition, emphasize an important role of ADP accumulation in the myofibrillar compartment, if the rephosphorylation of ADP by bound CK is not possible. Indeed, the absence of PCr or its presence at a too low concentration is an absolute prerequisite for rigor tension development at cytosolic [MgATP] higher than 
30 µM. During ischaemic stress, the depletion of PCr occurs very rapidly, thus favouring a decrease in the local MgATP/MgADP ratio. Our data show that at external [MgATP] and [MgADP] of 100 and 250 µM respectively, a marked rigor force even appears at millimolar PCr concentrations.
In conclusion, our data suggest that the local accumulation of ADP in myofibrils due to a fall in cellular PCr and inability of myofibrillar CK to rephosphorylate ADP produced by myosin ATPase is an important mechanism of diastolic tension rise under ischaemic conditions. In myofilaments, a loss of the CK control over the intracellular concentrations of adenine nucleotides may result in the appearance of marked subcellular gradients of MgATP and MgADP.
Time for primary review 35 days.
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Acknowledgements |
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This study was supported by an INSERM fellowship and ‘Fondation pour la Recherche Médicale’ (Dr. Veksler), Centre National de la Recherche Scientifique (Dr. Ventura-Clapier), and in part by a grant from the ‘Association Française contre les Myopathies’ and ‘Fondation de France’. The authors wish to acknowledge P. Mateo, F. Doucet-Levèbre and I. Ribaud for preparation of the cells. We thank Dr. H.C. Hartzell for helpful advice, Dr. X. Bigard for discussion of the results, Dr. E. Boehm for careful reading of the manuscript and Dr. R. Fischmeister for continuous support.
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