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Cardiovascular Research 1999 44(3):556-567; doi:10.1016/S0008-6363(99)00233-3
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

On the mechanism of the failure of mitochondrial function in isolated guinea-pig myocytes subjected to a Ca2+ overload

Kazunobu Ban*, Shunnosuke Handa and Reg A Chapman{dagger}

Division of Cardiology, Department of Medicine, School of Medicine, Tokai University, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan

* Corresponding author. Tel.: +81-463-931-121; fax: +81-463-936-679 ban{at}is.icc.u-tokai.ac.jp

Received 7 May 1999; accepted 20 July 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The influence of agents that inhibit the movement of Ca2+ across the mitochondrial membrane or Ca2+ dependent changes to this membrane upon the response of isolated ventricular myocytes to a Ca2+ overload has been investigated. Methods: The changes of intracellular Ca2+ and Mg2+ ([Ca2+]i, [Mg2+]i) (as reflected by cellular ATP), mitochondrial membrane potential ({Psi}m) and NADH was measured upon the response of isolated ventricular myocytes to a Ca2+ overload. Results: A slow depolarization of {Psi}m during Ca2+ depletion and its prompt recovery on Ca2+ repletion were unaffected by ruthenium red, clonazepam, CGP-37157 which is a high potent inhibitor of the mitochondrial Na+/Ca2+ antiport or cyclosporin A but a large delayed sustained depolarization was inhibited. The slow small fall in [Mg2+]i on Ca2+ depletion and a rapid recovery on Ca2+ repletion were unaffected by ruthenium red, clonazepam, CGP-37157 or cyclosporin A. A delayed sustained larger rise in [Mg2+]i was inhibited. The marked sustained fall in NADH autofluorescence that occurs on Ca2+ overload was attenuated and transient in the presence of ruthenium red, CGP-37157 and cyclosporin A. Conclusion: These results are consistent with an increase in Ca2+ cycling across the mitochondrial membrane provoked by the combined Na+ and Ca2+ overload of cardiac myocytes, causing a depolarization sufficient to uncouple respiration and lead to the depletion of cellular ATP.

KEYWORDS Calcium (cellular); Mitochondria; Myocytes

This article is referred to in the Editorial by J.J. Lemasters (pages 470–473) in this issue.


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Removal of Ca2+ from the medium bathing cardiac tissue followed by return to normal physiological fluid provokes a marked Ca2+ overload of the sarcoplasm, hypercontracture, fall in energy-rich phosphates and finally disruption of the cell membrane — the calcium paradox [4,6,23,27,31,40]. As these changes are similar to those provoked during and following anoxia or ischaemia, the calcium paradox has been used as a model to study Ca2+ overload damage. Initially work focused on the changes that occurred during the period of Ca2+ depletion to predispose the heart to Ca2+ overload. This identified the importance of a Na+ overload, that developed because the L-type Ca2+ channels conduct a sustained inward Na+ current. The discovery of an inhibitory action of Mg2+ led to the use of Ca2+ and Mg2+ free solutions to produce a larger and more consistent Na+ overload. When Ca2+ is returned to the bathing medium a Ca2+ overload is produced by a Ca2+ influx via sarcolemmal Na+/Ca2+ facilitated by the high intracellular Na+ ([Na+]i) [9,18,31,33]. More recently, the use of intracellular fluorescent indicators has confirmed that changes in intracellular Ca2+ ([Ca2+]i), Na+ and pH similar to those seen in multicellular preparations occur in isolated ventricular myocytes. Moreover, it has allowed the changes in mitochondrial function to be determined [27]. Removal of Ca2+ and Mg2+ from the bathing fluid results in either a small rise or little change in mitochondrial NADH and a small slow depolarization of mitochondrial membrane potential ({Psi}m) suggesting, consistent with early reports of a reduced oxygen consumption and elevated cellular ATP, that there is a fall in the demand for energy [6,34]. On return to normal physiological medium, the resulting Ca2+ overload provokes a marked and sustained fall in both {Psi}m and mitochondrial NADH. These changes, which are consistent with an uncoupling of respiration, were considered to act together with the increased energy demand to deplete energy rich phosphates. Two mechanisms were suggested for the depolarization of the mitochondrial membrane. One supposed that the combined Na+ and Ca2+ overload of the sarcoplasm resulted in increased Ca2+ cycling across the mitochondrial membrane which would cause a depolarization because the inward movement of charge associated with the entry of Ca2+ is not balanced by its efflux via the electrically neutral Na+/Ca2+ antiport (Fig. 1). The other supposed that the accumulation of Ca2+ within the mitochondrial matrix opened the large Ca2+ activated pore. The former mechanism was favored because the fall in mitochondrial NADH induced by Ca2+ overload is reversed when respiration is blocked by cyanide or rotenone [27]. The work to be reported below is an attempt to distinguish between these two alternatives and to determine the timing and degree of changes in cellular ATP (as reflected by changes in intracellular Mg2+ ([Mg2+]i)), [Ca2+]i, and {Psi}m in single ventricular myocytes. Part of this work has already appeared in abstract [3].


Figure 1
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  		Fig. 1 Schema of the Ca2+ cycling across the mitochondrial membrane and the sites of block by ruthenium red, clonazepam, CGP-37157 and cyclosporin A. Ruthenium red inhibits the Ca2+ uniport and CGP-37157 blocks the Na+/Ca2+ antiport. Cyclosporin A blocks the opening of the cyclosporin A sensitive pore, and it would also block the Na+/Ca2+ antiport on the mitochondria. The increase of Ca2+ cycling across the mitochondrial membrane on Ca2+ overload induces uncoupling of respiration from ATP synthesis.

 

   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
The experimental protocol was approved by the Experimental Committee of Bristol University, which confirms with the Report of the AVMA panel on Euthanasia in 1993 [2].

2.1 Cardiac myocytes isolation
Adult male guinea pigs (200–250 g) were killed by cervical dislocation and their hearts rapidly removed. Ventricular myocytes were isolated as described previously [31] except that 40 mM taurine was present in the medium into which the myocytes were finally dispersed. Then, myocytes were allowed to settle on a glass cover slip which formed the floor of a temperature controlled dish (volume 0.3 ml).

2.2 Experimental procedure
A cover slip with myocytes was placed under the inverted microscope (Nikon, Tokyo, Japan) and superfused with Tyrode solution containing (in mM): 140 NaCl, 2.5 KCl, 2.5 KOH, 2 CaCl2, 1 MgCl2, 5 sodium pyruvate, 5 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES) with a pH of 7.4, at a rate of 1 ml/min at 37°C. The Ca2+ depleting Tyrode solution was made by omitting CaCl2 (and in most cases MgCl2) and a –log10[Ca2+] (pCa) of 8.2 was achieved by adding 0.19 mM Ca-EGTA and 3.81 mM Tris–EGTA. The myocytes were superfused with normal Tyrode for 10 min, then by a solution containing no added Mg2+ and a pCa buffered to 8.2 for 10 min, then returned to normal Tyrode solution for a further 10 min. The epifluorescence of isolated myocytes was measured using a photon-counting system (Newcastle Photometric Systems, Newcastle-upon-Tyne, UK) as described previously [27]. At the end of an experiment the status of the respiratory chain was determined by recording the changes induced by 5 µM of the respiratory uncoupler carbocyanide m-chlorophenylhydrazone (CCCP). In experiments where NADH was measured, 5 mM CN– was used to inhibit respiration. Only data from viable myocytes, established by employing a Trypan Blue exclusion test at the end of experiment, were assessed. Throughout the experiment the image of the myocyte under study was viewed under red light with a video camera attached to the eyepiece of the microscope. The image was recorded on videotape and changes in cell length were measured.

2.3 Measurement of [Ca2+]i by Fura-2 fluorescence
Changes in [Ca2+]i were determined from the change in Fura-2 fluorescence ratio (excited at 350 and 380 nm with a 510 nm band-pass emission filter). Myocytes were preloaded by incubation in 5 µM of the acetomethyl ester of Fura-2 for 30 min at 37°C [27]. An approximate calibration was made in vitro as described previously [26].

Measurement of {Psi}m by JC-I fluorescence

To determine the changes in {Psi}m isolated myocytes were loaded by incubation in the presence of 5 µM 5,5',6,6'-tetrachloro-1,l',3, 3'-tetraethylbenzimidazolocarbo-cyanine iodide (JC-1; Molecular Probes, Eugene, OR, USA) for 15–20 min at 37°C [27,36] and the cell was excited at 490 nm and fluorescence was monitored at 530 nm and 590 nm. JC-1 is taken by mitochondria in proportion to the {Psi}m because it is lipophilic with delocalized positive charge and has been shown to have a high signal to noise ratio which correlates qualitatively with {Psi}m [16,27,30].

2.4 Measurement of NADH
NADH autofluorescence was excited at 350 nm and the emission measured between 420 and 540 nm as described previously [27].

2.5 Measurement of [Mg2+]i by Mag-Fura-2 fluorescence
[Mg2+]i was measured using the fluorescent probe Mag-Fura-2 [29]. Cells were loaded with 2.5 µM acetomethyl ester (Mag-Fura-2 AM: Molecular Probes) for 30 min at 37°C. Mag-Fura-2 was excited at 350 nm and 380 nm and the emission filter was a 510 nm band pass. An in vitro calibration was made using 2.5 µM Mag-Fura-2 and a varying [Mg2+] in a solution containing 150 mM KCl, 10 mM HEPES–KOH (pH 7.4) and 0.1 mM EGTA. The resulting data were fit by the equation:

Formula
where: –R is the 350/380 nm ratio at a given [Mg2+]i, Rmin and Rmax are the fluorescence ratio values under Mg2+-free and -saturating conditions, respectively and Sf2/Sb2 is the fluorescence ratio value for Mg2+-bound/Mg2+-free at 380 nm and Kd is the binding constant for Mg2+ [22]. [Mg2+]i is little affected by changes in the composition of the bathing ionic medium, but significant changes in [Mg2+]i occur when intracellular ATP is hydrolyzed (ATP being the major sink for [Mg2+]i) and [Mg2+]i therefore has been used as an indirect measure of cellular ATP levels [7,28,35].

2.6 Materials
CGP-37157 was provided by Dr. Alain DePover from Ciba Geigy (Basal, Switzerland), Clonazepam by Hoffmann La Roche (UK): both were dissolved in dimethyl sulfoxide to produce stock solutions of 40 mM and 1 mM respectively. Cyclosporin A was provided by Dr. Andrew Halestrap, Dept. of Biochemistry, University of Bristol. All other chemicals were purchased from Sigma (Poole, Dorset, UK).

2.7 Analysis and statistics
The epifluorescence as measured by photomultipliers, was digitized and stored on a microcomputer as counts per 0.2 s. Mathematical manipulation of files and the production of figures were done using the software package Fig.P (BioSoft, Cambridge, UK). To ease transfer, large data files were reduced by averaging up to ten successive points. Data were routinely compared using an analysis of variance and a Kurshal–Wallis test and where observations were made on the same myocyte a paired t-test was also applied (Statview 4.1, Abacus, Berkeley, CA, USA) on a Power Macintosh.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effects on the contractile response to Ca2+ overload
With Ca2+ overload, 87% of myocytes developed a strong sustained hypercontracture in the absence of an agent to affect mitochondrial function. The incidence of hypercontracture was unaffected by the presence of 0.2 µM cyclosporin A in the bathing medium throughout the experiment (87.5%) but was reduced to 78% by clonazepam (10–70 µM) and to 70% by CGP-37157 (1–5 µM) (P>0.05). When exposure to ruthenium red (2.5 µg/ml) was restricted to the last 2 min of Ca2+depletion, the incidence of hypercontracture was 65% (P<0.05).

3.2 The changes in [Ca2+]i on Ca2+ overload
Fig. 2 shows representative responses of [Ca2+]i and cell length to Ca2+ overload in the absence and presence of ruthenium red, CGP-37157 or cyclosporin A. A mean resting value of [Ca2+]i of 139±9 nM (n=7) was unaffected by the presence of 2.5 µg/ml ruthenium red, 40 µM clonazepam, 3 µM CGP-37157 or 0.2 µM cyclosporin A (P>0.05). On exposure to Tyrode solution free of Mg2+ and with a pCa buffered to 8.2, [Ca2+]i fell to 108±15 nM after 10 min. A similar value was reached in the presence of either ruthenium red, clonazepam, CGP-37157 or cyclosporin A (P>0.05). The peak value of [Ca2+]i in myocytes that developed a strong hypercontracture on Ca2+ repletion (1.1±0.1 µM; n=14) was unaffected by the presence of either 2.5 µg/ml ruthenium red (1.06±0.1 µM; n=6; P>0.05), 0.2 µM cyclosporin A (0.98±0.15 µM; n=5; P>0.05) or 3 µM CGP-37157 (0.87±0.12 µM; n=5; P>0.05). In the majority (67%) of myocytes, [Ca2+]i recovered to stabilize at a value above the original resting value over the next 5 min, while in the minority the recovery was slower and less complete.


Figure 2
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  		Fig. 2 The development of Ca2+ overload, as indicated by the changes in [Ca2+]i (———) and the changes in cell length (- - -), in the absence and presence of agents that change mitochondrial function: (A) in the absence of an agent to change mitochondrial function; (B) in the presence of ruthenium red; (C) in the presence of cyclosporin A; (D) in the presence of 3 µM CGP-37157. The timing of Ca2+ depletion and the presence of other agents in the bathing medium are indicated by the labeled horizontal bars.

 
3.3 The changes in {Psi}m
Fig. 3 shows representative responses of {Psi}m and cell length to Ca2+ overload in the absence and presence of ruthenium red, CGP-37157 or cyclosporin A. Consistent with previous work, the JC-1 fluorescence ratio showed a small steady rise during Ca2+ depletion, a change that was not affected by ruthenium red (0.25 or 2.5 µg/ml), CGP-37157 (1–3 µM), clonazepam (20–40 µM) or cyclosporin A (0.2 µM). Ca2+ repletion induced a marked hypercontracture, which was accompanied by an immediate rapid fall in the JC-1 ratio back to the original value. This was followed, after a delay of 40.6±11.5 s (n=10, P>0.01, compared to the time to peak of the hypercontracture or rise in [Ca2+]i) by a larger rise. This rise, once stabilized, was not further increased by exposure to CCCP, indicating that {Psi}m was fully depolarized (Fig. 3A). In myocytes, where hypercontracture developed promptly on return to Tyrode solution, the initial recovery in the JC-1 fluorescence ratio persisted in the presence of 0.25 µg/ml of ruthenium red but the subsequent rise was much delayed (271±35.1 s; n=8; a value significantly different from control P<0.002; Fig. 3B). When either 2.5 µg/ml ruthenium red, 5 µM CGP-37157 or 0.2 µM cyclosporin A were present, the prompt fall in JC-1 fluorescence ratio still accompanied the development of the hypercontracture but the delayed secondary rise was inhibited (Fig. 3D). In the presence of 1 µM CGP-37157 the subsequent rise was generally inhibited (Fig. 3C). Clonazepam, when present at 10 µM had little effect on the changes in JC-1 fluorescence ratio associated with Ca2+ overload, while at 30–50 µM the secondary rise was further delayed (143±55 s; n=5; P<0.05) or completely inhibited (n=4, data not shown). In the presence of ruthenium red, CGP-37157, clonazepam (data not shown) or cyclosporin A, where a delayed rise did not develop a large increase was induced by CCCP (Fig. 3C,D). When the JC-1 fluorescence ratio reached the plateau, CCCP had little effect (Fig. 3B). The changes in JC-1 fluorescence in five myocytes that developed on Ca2+ overload were unaffected by the presence of 2 µM oligomycin (data not shown).


Figure 3
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  		Fig. 3 The effect of altered mitochondrial function on the changes in mitochondrial membrane potential of isolated ventricular myocytes induced by Ca2+ overload: (———) JC-1 ratio; (- - -) cell length. (A) In the absence of an agent to alter mitochondrial function, a slow rise in JC-1 fluorescence (depolarization of {Psi}m) occurred during Ca2+ depletion which quickly recovered on Ca2+ repletion to be shortly followed by a large rise in fluorescence ratio. (B) In the presence of 0.25 µg/ml ruthenium red, the hypercontracture and associated recovery of JC-1 fluorescence ratio were unaffected but the delayed large rise in JC-1 fluorescence was further delayed. (C) In the presence 1 µM CGP-37157, the hypercontracture and changes in JC-1 fluorescence on Ca2+ repletion were unaffected with the exception of the large delayed increase which was inhibited. (D) In the presence of 0.2 µM cyclosporin A, the development of the hypercontracture and the changes in JC-1 fluorescence ratio on the induction of Ca2+ overload were unaffected with the exception of the delayed large rise which was inhibited. When the delayed rise in JC-1 fluorescence ratio developed (A and B) application of 5 µM CCCP had little effect on the JC-1 fluorescence ratio, however when this increase was inhibited (C and D) the application of CCCP provoked a large increase. The periods of change in the composition of the bathing medium are indicated by the labeled horizontal bars; time is from the onset of the experiment.

 
3.4 The changes in NADH autofluorescence
Fig. 4 shows representative responses of NADH autofluorescence and cell length to Ca2+ overload in the absence and the presence of the ruthenium red, CGP-37157 or cyclosporin A. NADH autofluorescence of myocytes was little changed during a 10 min exposure to Tyrode solution free of Mg2+ and with the pCa buffered to 8.2 and was unaffected by the presence at ruthenium red, CGP-37157 or cyclosporin A (Fig. 4). A steep maintained fall in NADH autofluorescence was seen in myocytes that developed a hypercontracture on Ca2+ repletion (relative NADH autofluorescence fell to 29.0±6.2% of the pre-Ca2+ repletion value; n=9). In myocytes that developed a strong hypercontracture, this decrease in the relative NADH autofluorescence was significantly slowed and attenuated by 2.5 µg/ml ruthenium red (relative autofluorescence fell to nadir of 58.7±8.9%; n=9; P<0.05), 3 µM CGP-37157 (60.2±8.0%; n=7; P<0.05) or 0.2 µM cyclosporin A (55.8±12.2%; n=7; P<0.05) and under these conditions the NADH autofluorescence recovered often with a sight overshoot (Fig. 4).


Figure 4
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  		Fig. 4 The change in relative NADH autofluorescence on initiation of Ca2+ overload in the presence and absence of agents that affect mitochondrial function: (———) NADH autofluorescence; (- - -) cell length. The changes observed when Ca2+ overload is induced in the absence of an agent that alter mitochondrial function (A) are compared to those seen in the presence of 2.5 µg/ml ruthenium red, added 2 min before Ca2+ repletion (B), 1 µM CGP-37157 present throughout Ca2+ depletion and repletion (C) and 0.2 µM cyclosporin A present throughout Ca2+ depletion and repletion (D). The solid labeled horizontal lines indicate the periods when composition of the bathing medium was changed; time is from the onset of the experiment.

 
3.5 The changes in [Mg2+]i
Fig. 5 shows representative responses of [Mg2+]i and cell length to Ca2+ overload in the absence and the presence of ruthenium red, CGP37157 or cyclosporin A. The resting Mag-Fura-2 fluorescence ratio was equivalent to a [Mg2+]i of 0.66±0.02 mM (n=54) at 37°C in quiescent myocytes bathed by normal Tyrode solution. A slow fall in Mag-Fura-2 fluorescence ratio, equivalent to a change in [Mg2+]i from 0.67±0.08 mM to 0.37±0.07 mM after 10 min, was seen in myocytes exposed to Tyrode solution with pCa buffered to 8.2 without added Mg2+ (n=11; P<0.0001; Fig. 5A). The mean values for [Mg2+]i after 10 min of Ca2+ depletion were not significantly different from control (P>0.1) when either ruthenium red, CGP-37157 or cyclosporin A, clonazepam or 1 mM Mg2+ were present.


Figure 5
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  		Fig. 5 Changes in Mag-Fura-2 fluorescence ratio subjected to Ca2+ overload and the associated changes in the absence and presence of agents that affect mitochondrial function: (———) [Mg2+]i; (- - -) cell length. (A) A complete typical control experiment done in the absence of a mitochondrial inhibitor. During Ca2+ depletion a small slow fall in Mag-Fura-2 fluorescence ratio occurred; on Ca2+ repletion an initial small rise accompanied hypercontracture and, after a delay, a large sustained rise in fluorescence ratio developed which was not further affected by the application of 5 µM CCCP. (B) Part of an experiment done to show the changes observed when Ca2+ repletion was made after 10 min Ca2+ depletion in the presence of 2.5 µg/ml ruthenium red. The hypercontracture and associated rise in fluorescence ratio were unaffected while a delayed large rise in fluorescence ratio failed to develop. (C) Part of an experiment done in the continued presence of 1 µM CGP-37157, the hypercontracture and associated rise in fluorescence ratio on Ca2+ repletion were unaffected but the delayed rise was inhibited. (D) Part of an experiment done in the continued presence of 0.2 µM cyclosporin A, where the hypercontracture and associated change in fluorescence ratio on Ca2+ repletion were unaffected but the delayed rise was inhibited. When the delayed sustained rise in fluorescence ratio failed to develop (B, C and D) subsequent application of 5 µM CCCP provoked a large rise. The labeled horizontal bars indicate when the periods when the composition of the bathing medium was changed; time is from the onset of the experiment.

 
A rapid increase in the Mag-Fura-2 ratio coincident with the hypercontracture occurred. After a delay of 90.8±4.9 s (n=10), a larger and sustained increase in Mag-Fura-2 fluorescence ratio was observed which was not affected by the application of CCCP once stabilized and reached a value equivalent to a [Mg2+]i of 2.85±0.40 mM (Fig. 5A and Fig. 6A). The timing and amplitude of the initial change in Mag-Fura-2 fluorescence ratio was unaffected by ruthenium red, CGP-37157, cyclosporin A or clonazepam (P>0.1) while the secondary rise generally either failed to occur (Fig. 5B,C,D) or was much delayed (mean delays were: 179±50 s for 2.5 µg/ml ruthenium red (n=3), 167±30 s for 3 µM CGP-37157 (n=3), 388±17 s for 40 µM clonazepam (n=2) and 300 s for 0.2 µM cyclosporin A (n=1)). The mean values for Mag-Fura-2 fluorescence ratio (and the calculated [Mg2+]i) 10 min after return to normal were significantly reduced when Ca2+ repletion was made in the presence of either ruthenium red, CGP-37157, clonazepam or cyclosporin A (mean values were: 0.68±0.13 mM for ruthenium red (n=9), 1.00±0.13 mM for CGP-37157 (n=8), 0.65±0.08 mM for clonazepam (n=3) and 0.90±0.35 mM for cyclosporin A (n=5), Fig. 6A; P<0.01)). In those myocytes where a large delayed rise in Mag-Fura-2 ratio failed to develop, application of CCCP produced a large and sustained increase in Mag-Fura-2 fluorescence ratio (Figs. 5 and 6Go). The rise in Mag-Fura-2 ratio, on application of CCCP, was rapid in the presence of ruthenium red but noticeably slower and often developed in two stages in the presence of CGP-37157, clonazepam (data not shown) or cyclosporin A (Fig. 5). The final values of Mag-Fura-2 fluorescence ratio or the calculated [Mg2+]i were somewhat smaller than the value reached 10 min after return to normal Tyrode solution in the control experiments but achieved statistical significance only in the case of cyclosporin A (1.33±0.31 mM; n=5; P<0.05; Fig. 6B). The changes in Mag-Fura-2 fluorescence ratio that were seen when Ca2+ repletion was made in the continued absence of extracellular Mg2+ were not different from those in which 1 mM Mg2+ was present at Ca2+ repletion (data not shown).


Figure 6
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  		Fig. 6 (A) The estimated values (mean±SEM) of [Mg2+]i, reached 10 min after the induction of a Ca2+ overload. The values for 2.5 µg/ml ruthenium red, 3 µM CGP-37157, 30 µM clonazepam and 0.2 µM cyclosporin A are significantly reduced when compared to the change seen in the absence of these agents (P<0.01). (B) The estimated values (mean±SEM) of [Mg2+] i, reached when 5 µM CCCP was applied 10 min after the induction of a Ca2+ overload. Although the values reached in the presence of ruthenium red, CGP-37157, clonazepam and cyclosporin A are reduced compared to control, these differences achieve statistical significance (P<0.05) only in the case of cyclosporin A. cont, control; R.R, ruthenium red; CGP, CGP-37157; CLZ, clonazepam; CyA, cyclosporin A.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
4.1 The effects of inhibitors
The effects of inhibitors of Ca2+ influx and efflux across the mitochondrial membrane and of the Ca2+ dependent pore on the changes provoked by Ca2+ overload have been determined. Ruthenium red was chosen because it is a potent inhibitor of the Ca2+ uniporter in the mitochondrial membrane [32,37] without effect on the sarcolemmal Na+ dependent Ca2+ uptake at concentrations of up to 25 µM [20]. However, it can affect the Ca2+ channels, the binding of Ca2+ to the sarcolemma and Ca2+-ATPases in the sarcoplasmic reticulum (SR) and has been reported to protect the heart from ischaemia or the oxidant stress [17,20,38]. The benzodiazepines and benzothiazepines block the mitochondrial Na+/Ca2+ antiport without effect on either the mitochondrial Ca2+ uptake, the sarcolemmal Na+/Ca2+ exchange or Na+ pump or the Ca2+-ATPase in the SR [13]. Clonazepam, unlike diltiazem, does not inhibit currents through L-type voltage-dependent Ca2+ channels [12,24,25]. A novel benzothiazepine, CGP-37157 exhibits a high potency (IC50 0.4 µM) for the mitochondrial Na+/Ca2+ antiport and is also without effect on L-type Ca2+ channels [11–13,24]. Although cyclosporin A was used to block the cyclosporin A sensitive pore on the mitochondrial membrane, specific caution was made for its other effects [5,14,19,21,38].

4.2 The changes in [Ca2+]i, NADH autofluorescence, [Mg2+]i and {Psi}m during Ca2+ depletion
We have shown that, during exposure to Tyrode solution free of Ca2+ and Mg2+, there is a small sustained fall in [Ca2+]i, a slow increase in JC-1 fluorescence ratio (indicative of a depolarization of {Psi}m), and little change or a small rise in NADH autofluorescence. The resting value of [Mg2+]i is similar to previously reported values for mammalian heart cells [7,22,29,35,39]. However, because Mag-Fura-2 shows some sensitivity to Ca2+ and pH, this has to be taken into account when estimating [Mg2+]i whenever the intracellular levels of these cations change. A fall in Mag-Fura-2 fluorescence ratio accompanies Ca2+ depletion and is probably due to a fall in [Mg2+]i because the changes in [Ca2+]i or pHi are too small and over a range that does not affect the signal [7,27]. This means that [Mg2+]i falls to half its resting level during a 10 min period of perfusion with Tyrode solution free of Ca2+. As this fall persists when Ca2+ depletion is performed in the presence of 1 mM Mg2+, it is unlikely to be associated with the movement of Mg2+ across the cell membrane and most probably reflects the known rise in ATP [6,34]. The changes in [Ca2+]i, {Psi}m, NADH and [Mg2+]i, associated with perfusion of isolated cardiac myocytes with Tyrode solution free of divalent cations are unaffected either by block of the mitochondrial Ca2+ uniport or the mitochondrial Na+/Ca2+ exchange or by the presence of cyclosporin A. These observations suggest that the effect on {Psi}m is not mediated by changed intramitochondrial [Ca2+] or its effect on intramitochondrial dehydrogenases [15] and probably results from the effect of the rise in ATP feeding back on the respiratory chain.

4.3 The effects of inhibitors for hypercontracture during Ca2+ repletion
Firstly, we showed that hypercontracture can be reliably used as an indication of Ca2+ overload when other parameters are measured by performing experiments with various agents upon Ca2+ overload. Then the recovery of the JC-1 fluorescence ratio and the rise in Mag-Fura-2 fluorescence ratio were evaluated in relation with the onset of the hypercontracture, which are unaffected by ruthenium red, clonazepam, CGP-37157 or cyclosporin A. These changes are therefore unlikely to be mediated by Ca2+ accumulation within the mitochondria so that a reactivation of respiration due to increased energy demand would seem a plausible mechanism: an idea consistent with the persistence of a small fall in NADH in the presence of ruthenium red (Fig. 3B).

4.4 Role of [Mg2+]i during Ca2+ repletion
The data for the sensitivity of Mag-Fura-2 to pH and [Ca2+]i provided by Buri et al. [7] suggest that the transient acidification and rise in [Ca2+]i that occur on Ca2+ repletion will affect the fluorescence of the probe and contribute to the rise in Mag-Fura-2 fluorescence ratio. However, the rise in [Mg2+]i would seem to be significant because the interference due to the measured changes in pHi and [Ca2+]i is unlikely to contribute more than 20% to the change in Mag-Fura-2 fluorescence ratio and the recovery of the Mag-Fura-2 ratio is much slower than that of either pHi or [Ca2+]i (compare Fig. 5 with Fig. 2 and Fig. 1D in Ref. [27]). This initial rapid increase in Mag-Fura-2 ratio persists when Ca2+ repletion is made in the absence of bathing Mg2+ and probably doses not involve an influx of Mg2+ into the cell. It most probably reflects a fall in ATP associated with an increased energy demand, a notion supported by the failure of ruthenium red, clonazepam and CGP-37157 to affect this initial change in Mag-Fura-2 fluorescence and to inhibit completely the initial fall in NADH (Fig. 4). The sustained hypercontracture and activation of the Ca2+-ATPases within the cell will increase ATP consumption to result in a rise in [Mg2+]i.

By the time the large delayed rise in Mag-Fura-2 fluorescence ratio develops, [Ca2+]i and pHi will have fallen back to values where the interference of these ions on the signal is small. The large change in Mag-Fura-2 fluorescence ratio therefore indicates an unambiguous rise in [Mg2+]i. The fact that the rise in [Mg2+]i is unaffected when Ca2+ repletion is made in the absence of extracellular Mg2+ suggests that movement of Mg2+ across the cell membrane is not involved. This rise in [Mg2+]i is consistent with a marked depletion of cellular ATP as the Mag-Fura-2 ratio is not further increased by the application of CCCP. This being so, the sequence of events on Ca2+ overload are: — hypercontracture, followed after a delay by depolarization of the mitochondrial membrane followed after a further delay by the near exhaustion of cellular ATP.

4.5 Evidence for the role of Ca2+ entry via the uniporter
The large delayed increase in JC-1 fluorescence develops significantly later than the peak of the change in [Ca2+]i, and reaches a value not further increased by CCCP and should therefore uncouple respiration from ATP production. The ability of ruthenium red to delay or prevent this depolarization (the uncoupling of respiration) and the rise in [Mg2+]i (fall in ATP) and allow the recovery of NADH in myocytes that develop hypercontracture on Ca2+ repletion, clearly implicates Ca2+ entry via the uniport in the development of these changes.

4.6 Evidence for the role of Ca2+ cycling in hypercontracture
The ability of clonazepam and CGP-37157 to block the delayed rise in JC-1 fluorescence ratio favors the idea that increased Ca2+ cycling across the mitochondrial membrane is the mechanism responsible for the depolarization of {Psi}m and the consequent changes rather than the accumulation of Ca2+ within the mitochondria. This is because inhibition of the mitochondrial Na+/Ca2+ should prevent Ca2+ efflux to augment the rise in mitochondria1 [Ca2+] and thereby increase the likelihood of opening of the cyclosporin A sensitive pore. However, clonazepam and CGP-37157 inhibit the large delayed rise in JC-1 fluorescence ratio in Ca2+ overloaded myocytes (but not the depolarization induced by CCCP) showing that this depolarization is prevented when Ca2+ cycling but not Ca2+ accumulation within the mitochondria is blocked. Furthermore, the inhibition of the secondary rise in [Mg2+]i, the attenuation of the fall in NADH autofluorescence and the later recovery of NADH levels suggest that respiration can meet the increased energy demand in the Ca2+ overloaded cardiac myocyte. This means that the uncoupling of respiration is critical in causing the fall in cellular ATP which is the consequence of Ca2+ overload.

The effects of the inhibitors of the mitochondrial Na+/Ca2+ antiport would seem to rule out the activation of the cyclosporin sensitive pore during Ca2+ overload; a conclusion that is at variance with the observed effects of cyclosporin A. Clearly, the effects of cyclosporin A and/or the benzothiazepines and benzodiazepines cannot be as specific as formerly believed. A shared ability to inhibit adenylate translocase could explain the maintenance of {Psi}m but not the failure of ATP to fall (especially as the metabolic substrate is pyruvate), or the absence of an effect of oligomycin.

Unpublished work on isolated cardiac mitochondria has failed to detect an effect of CGP-37157 on the cyclosporin A sensitive pore (Ban, Connern, Halestrap and Chapman), however, cyclosporin A has been reported to inhibit the Na+/Ca2+ antiport [1]. These findings suggest that not only ruthenium red, CGP-37157 and clonazepam but also cyclosporin A can block Ca2+ cycling across the mitochondrial membrane. This would mean that increased Ca2+ cycling is responsible for the depolarization of {Psi}m and the consequent depletion of energy-rich phosphates and mitochondrial NADH. Evidence of a similarity in action of clonazepam, CGP-37157 and cyclosporin A are suggested by the slower rise of [Mg2+]i provoked by application of CCCP, however, additional effects of cyclosporin A are indicated by the reduced rise in [Mg2+]i under these conditions (Fig. 5 and Fig. 6B).

4.7 The point of no return
The changes in mitochondrial function, as opposed to the disruption of the ionic balance across the cell membrane, may be "the point of no return" along the path to myocyte death induced by Ca2+ overload. This is because once ATP levels become depleted, the cell will lose the ability to reduce the Na+ overload and thereby the Ca2+ overload or to expel H+. These effects may be important in the disruption of cellular function whenever [Na+]i and [Ca2+]i rise because elevated [Ca2+]i, reduced pHi and ATP levels are synergistic in activating a range of cellular phospholipases and proteases. The activation of these enzymes would be expected to cause disruption of the cytoskeleton, intracellular organelles and the cell membrane [10]. Furthermore, because of the close interrelationship between [Na+]i and [Ca2+]i by way of the sarcolemmal and mitochondrial Na+/Ca2+ exchangers, effects on ATP production from respiration may be compromised by increased Ca2+ cycling across the mitochondrial membrane during other cellular disturbances. The inhibition of respiration during ischaemia and anoxia would be reinforced by this mechanism and on reperfusion the influence of an increased Ca2+ cycling across the mitochondrial membrane would hinder the recovery of ATP levels. A scheme of this sort could explain at least part of the effectiveness of ruthenium red and even cyclosporin A to reduce protein loss from perfused hearts and the depletion of ATP under these conditions [8,17,19,38].

Time for primary review 36 days.


   Acknowledgements
 
The technical help of Mrs. Valerie Buswell is gratefully acknowledged. We also wish to thank Dr. Krai Chatamra for help making the figures and Dr. Phillip Dooley for comments on the manuscript.


   Notes
 
{dagger} Deceased. Back


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

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