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Cardiovascular Research 2005 67(2):291-300; doi:10.1016/j.cardiores.2005.03.015
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Copyright © 2005, European Society of Cardiology

Role of mitochondrial re-energization and Ca2+ influx in reperfusion injury of metabolically inhibited cardiac myocytes

Glenn C. Rodrigo* and Nicholas B. Standen

Department of Cell Physiology and Pharmacology, University of Leicester, PO Box 138, Leicester LE1 9HN, UK

* Corresponding author. Current address: Department of Cardiovascular Sciences, University of Leicester, Clinical Sciences Wing, Glenfield General Hospital, Leicester LE3 9QP, UK. Tel.: +44 116 256 3028; fax: +44 116 287 5792. Email address: gcr4{at}le.ac.uk

Received 20 December 2004; revised 9 March 2005; accepted 21 March 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Data Supplement
 References
 
Objective: We used isolated myocytes to investigate the role of mitochondrial re-energization and Ca2+ influx during reperfusion on hypercontracture, loss of Ca2+ homeostasis and contractile function.

Methods: Isolated adult rat ventricular myocytes were exposed to metabolic inhibition (NaCN and iodoacetate) and reperfusion injury was assessed from hypercontracture, loss of Ca2+ homeostasis ([Ca2+]i measured with fura-2) and failure of contraction in response to electrical stimulation. Mitochondrial membrane potential was followed using the potentiometric dye tetramethylrhodamine ethyl ester.

Results: Metabolic inhibition led to contractile failure and rigor accompanied by a sustained increase in [Ca2+]i. Reperfusion after 10 min metabolic inhibition led to an abrupt repolarization of the mitochondrial membrane potential (after 25.5 ± 1.2 s), a transient fall in [Ca2+]i followed by an abrupt hypercontracture (37.1 ± 1.8 s) in 84% of myocytes. Ca2+ homeostasis (diastolic [Ca2+]i<250 nM) recovered in only 23.3 ± 5.1% of cells and contractions recovered in 15.3 ± 2.2%. Oligomycin abolished the hypercontracture on reperfusion, but mitochondrial repolarization was unaffected. Preventing Ca2+ influx during reperfusion with Ca2+-free Tyrode or with an inhibitor of Na+/Ca2+ exchange did not prevent the hypercontracture, but increased the percentage of cells recovering Ca2+ homeostasis and contractile function. The presence of 0.5 µM cyclosporin A did not prevent hypercontracture but increased the percentage of cells recovering Ca2+ homeostasis to 56.2 ± 3.6% and contractile function to 52 ± 4.3%.

Conclusions: Reperfusion-induced hypercontracture, and loss of Ca2+ homeostasis and contractile function are initiated following mitochondrial re-energization. The hypercontracture requires the production of oxidative ATP but not Ca2+ influx during reperfusion. Loss of Ca2+ homeostasis and contractile function are linked to Ca2+ influx during reperfusion, probably via opening of mitochondrial permeability transition pores.

KEYWORDS Reperfusion; Myocytes; Mitochondria; Na+/Ca2+ exchanger; Calcium; Cardiac muscle; Ischaemia


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Data Supplement
 References
 
Reperfusion injury of ischaemic myocardium is characterized by the appearance of contraction band necrosis, loss of ionic homeostasis, depletion of high energy phosphates and release of intracellular enzymes. This injury is thought to result from a combination of severe hypercontracture, which in the intact heart gives rise to mechanical damage of cells weakened by the preceding ischaemia [1,2], and disruption of mitochondrial function resulting in depleted levels of ATP [3].

Reperfusion of ischaemic myocardium leads to increased availability of ATP which, coupled to raised [Ca2+]i, is thought to initiate a strong hypercontracture [4–6]. Reoxygenation of cardiomyocytes leads to sequestration of excess Ca2+ by the sarcoplasmic reticulum, which together with Ca2+ influx via reverse-mode Na+/Ca2+ exchange (NCX) generates large Ca2+ oscillations resulting from sarcoplasmic reticulum release and uptake precipitating cell hypercontracture [1,7]. Assuming that oxidative phosphorylation is responsible for the recovery of ATP, rapid re-energization and repolarization of the mitochondrial membrane potential ({Delta}{Psi}m) will be key events in initiating the reperfusion-induced hypercontracture. Indeed, the presence of mitochondrial uncoupling agents at the time of reperfusion, which prevent mitochondrial re-energization, can reduce re-oxygenation injury of hypoxic myocardium [8]. In addition to mechanical injury originating from hypercontracture, reperfusion of ischaemic myocardium also causes mitochondrial permeability transition pores (MPTP) to form and open, which initiates biochemical injury [3,9]. Mitochondrial repolarization is a contributory factor in the formation and opening of MPTPs during reperfusion [10], together with reactive oxygen species (ROS) and high cytosolic Ca2+ driving mitochondrial Ca2+ accumulation [3]. Therefore, repolarization of {Delta}{Psi}m, which occurs within the first minute of reperfusion [11,12] may be the primary event that triggers both mechanical and biochemical injury.

However, we have shown that reperfusion of adult rat cardiac myocytes following complete metabolic inhibition results in a rapid hypercontracture (~20 s) that follows mitochondrial repolarization but develops before the reperfusion-induced increase in [Ca2+]i [13]. We have therefore investigated the role of mitochondrial re-energization in reperfusion injury of adult rat ventricular myocytes following complete MI and determined whether Ca2+ influx contributes directly to hypercontracture and/or loss of function. Our results show that mitochondrial repolarization precedes and probably initiates both hypercontracture and loss of contractile function and Ca2+ homeostasis. However, hypercontracture is linked primarily to increased availability of oxidative ATP secondary to mitochondrial repolarization and does not require Ca2+ influx on reperfusion. In contrast, loss of Ca2+ homeostasis and contractile function require Ca2+ influx and are ameliorated by cyclosporin A, suggesting that they are a consequence of MPTP formation in response to Ca2+ influx.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Appendix A. Data Supplement
 References
 
2.1. Isolation of single ventricular myocytes
Adult male Wistar rats (around 300 g) were killed by cervical dislocation and single ventricular myocytes isolated by enzymatic dissociation, as described previously [13]. The investigation conformed 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). Myocytes were stored at 15 °C and used on the day of isolation.

2.2. Measurement of contractile activity, mitochondrial membrane potential and intracellular ATP and Ca2+
Our methods for measurement of contraction, mitochondrial membrane potential ({Delta}{Psi}m), [Ca2+]i and [ATP]i have been described previously [11,13,14]. Briefly, myocytes were placed in a 300 µl chamber on the stage of an inverted microscope, continuously superfused with Tyrode solution at a rate of 5 ml/min, and stimulated at 1 Hz by electrical field stimulation. The washout time of the bath was under 10 s. Contractile activity of individual myocytes was determined from changes in cell length using a video edge-detection system (Crescent Electronics). To observe recovery of contractile function, cells were observed under bright-field illumination and contractile responses to electrical field stimulation determined.

To measure [Ca2+]i, myocytes were loaded with fura-2 and excited alternately at 340 and 380 nm with a monochromator with emitted light collected at >520 nm. Fluorescence intensity was measured from 8–16 cells simultaneously using a video-imaging system (Photon Technology International). Free calcium values were estimated using a look up table generated with a fura-2 imaging calibration kit (Molecular Probes) [14]. The degree of non-cytosolic loading of fura-2 and its contribution to the measured [Ca2+]i was determined by quenching the cytosolic signal with MnCl2 (see online data supplement). [Mg2+]i was estimated using the indicator Mg Green which was excited at 485 nm with emitted light >520 nm collected using a photomultiplier tube. {Delta}{Psi}m was monitored using the dye tetramethylrhodamine ethyl ester (TMRE) [11], illuminated at 475 nM for 33 ms at 0.1 Hz to limit photodamage with emitted light collected at >510 nm. Although the relationship between TMRE fluorescence and {Delta}{Psi}m cannot be calibrated directly in intact cells, we have shown previously that the change in TMRE fluorescence in response to MI is comparable to that observed during mitochondrial uncoupling with FCCP, which causes complete mitochondrial depolarization (~150 mV) [11].

2.3. Drugs and experimental solutions
Tyrode solution contained (mM): NaCl 135, KCl 5, NaH2PO4 0.33, Na-pyruvate 5, glucose 10, MgCl2 1, CaCl2 2, Hepes 10, titrated to pH 7.4 with NaOH. In Ca2+-free Tyrode CaCl2 was omitted and Ca2+ buffered with 1 mM EGTA. Metabolic inhibition was induced by superfusing the cells with metabolic inhibition (MI) Tyrode, which contained 2 mM NaCN and 1 mM iodoacetic acid (IAA) in substrate-free Tyrode. Cyclosporin A (Sigma) at 5 mM, sanglifehrin A (Novartis) at 1 mM and oligomycin (Sigma) at 10 mg.ml–1 were dissolved in DMSO. The final DMSO concentration in all experiments did not exceed 0.1%, which had no measurable effect on the cellular parameters measured. Myocytes were loaded with fura-2 (5 µM) and TMRE (6 µM) for 15 min, washed twice with normal Tyrode and left for 30 min prior to use. Fura-2 (5 mM) and TMRE (5 mM) (Molecular Probes Inc.) were dissolved in DMSO containing 5% pluronic acid.

2.4. Data acquisition and statistics
Statistical significance was calculated using Student's t-test, and p<0.05 was considered significant. Data are presented as mean ± S.E.M. For experiments involving measurements from a number of cells in a field of view, we have given numbers of experiments and cells as n = experiments; cells. Experiments were done at 35 °C.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Appendix A. Data Supplement
 References
 
3.1. Reperfusion after metabolic inhibition leads to rapid and abrupt hypercontracture and loss of Ca2+ homeostasis
Fig. 1A shows a typical recording of cell length from a rat ventricular myocyte exposed to metabolic inhibition using CN– and iodoacetate in substrate-free solution (MI Tyrode). In this and all subsequent experiments described in this paper, cells were stimulated at 1 Hz throughout using field stimulation. MI Tyrode led to contractile failure followed closely by cell shortening into rigor, indicating a collapse of ATP levels in the region of the contractile proteins. Shortly after reperfusion with normal Tyrode, which contained the metabolic substrates glucose and pyruvate, the myocyte shortened further into a hypercontracted state, characterized by loss of rectangular shape and striations and by membrane blebbing. Such behaviour is consistent with previous reports by others and ourselves [4,7,14,15].


Figure 1
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  		Fig. 1 Effect of metabolic inhibition and reperfusion on contraction and [Ca2+]i. (A) Recording of cell length from a single myocyte exposed to metabolic inhibition (MI Tyrode) for 10 min and reperfused with normal Tyrode and stimulated at 1 Hz throughout. (B) Bar chart showing the mean ± SEM percentage of cells which have hypercontracted and those which have recovered contractile activity in response to electrical stimulation after 10 min reperfusion with normal Tyrode (n = 20; 158). (C) Simultaneous recordings of [Ca2+]i from eleven myocytes within a single field of view during MI for 10 min and reperfusion with normal Tyrode for 10 min. Each trace is the [Ca2+]i recorded from a different cell using an imaging system. Images were sampled at 0.1 Hz and Ca2+ transients were evoked at 1 Hz resulting in aliasing of the calcium transients as they change phase with the sampled data points. (D) Bar chart showing the mean diastolic [Ca2+]i recorded in normal Tyrode (error bars for normal Tyrode are too small to show), at the end of 10 min MI, and after 10 min reperfusion with normal Tyrode, as indicated by the arrows in (C) (n = 43; 722). (E) Relationship between percentage of cells hypercontracted and the [Ca2+]i at the end of metabolic inhibition (n = 6–14 experiments).

 
In similar experiments where cells were exposed to MI Tyrode for 10 min, cell morphology was determined from fields of cells containing 6–10 cells at the end of a 10 min period of reperfusion with normal Tyrode. The mean data from such experiments show that over 80% of such cells hypercontracted while only about 15% recovered contractile function in response to electrical field stimulation (Fig. 1B). Fig. 1C shows a simultaneous recording of the fura-2 ratio from 11 myocytes during MI and reperfusion. [Ca2+]i increased progressively to 200–500 nM after 10 min in MI Tyrode and on reperfusion fell transiently before increasing steadily. The mean [Ca2+]i recorded from many such experiments is shown in Fig. 1D. Although [Ca2+]i at the end of MI differed between individual cells, Fig. 1E shows that the percentage of cells that hypercontracted on reperfusion was not significantly different between the [Ca2+]i ranges of 150–250, 250–500 and >500 nM.

3.2. Hypercontracture depends on mitochondrial re-energization
It has been suggested that hypercontracture results from increased availability of ATP resulting from rapid ATP synthesis following re-energization [1,4]. Fig. 2A shows simultaneous recordings of TMRE fluorescence, as an indicator of {Delta}{Psi}m, and cell length in a myocyte exposed to metabolic inhibition and reperfusion. {Delta}{Psi}m depolarized during MI, indicated by an increase in TMRE fluorescence, and recovered rapidly on reperfusion. It can be seen that mitochondrial repolarization preceded the development of hypercontracture, and this is also clear from the mean times to the onset and 90% completion of repolarization and hypercontracture shown in Fig. 2B and C.


Figure 2
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  		Fig. 2 Relationship between mitochondrial membrane potential and hypercontraction during metabolic inhibition and reperfusion. (A) Simultaneous recording of TMRE fluorescence (indicating changes in {Delta}{Psi}m) and cell length from a myocyte during MI and reperfusion. Under the loading conditions we used TMRE fluorescence is reduced by autoquenching when the dye is concentrated in mitochondria by the highly negative {Delta}{Psi}m. Mitochondrial depolarization leads to a redistribution of TMRE into the cytoplasm and a corresponding increase in fluorescence. (B) Mean times from the start of reperfusion to the onset of mitochondrial repolarization and the onset of hypercontracture as indicated. (C) Mean times to 90% completion of repolarization and hypercontracture. Results are from 8 cells in which {Delta}{Psi}m and length were recorded simultaneously. *p<0.01.

 
Repolarization of {Delta}{Psi}m restores the driving force for the F0/F1 ATP synthetase and the production of ATP. To investigate the time course of the change in intracellular ATP, we used the Mg2+-sensitive dye Mg Green. Intracellular free Mg2+ rises when ATP is hydrolysed, so that fluorescent Mg2+ indicators can give a sensitive measure of rates of change of ATP [16,17]. Fig. 3A shows an abrupt rise in Mg2+ about 100 s after application of MI, indicating the fall in ATP that led to rigor contraction. Shortly after reperfusion, Mg2+ fell sharply, indicating a rise in ATP that coincided with hypercontracture. To confirm the role of ATP synthesis in hypercontracture, we used the inhibitor of the mitochondrial F0/F1 ATP synthetase oligomycin (5 µg ml–1). Fig. 3B and C show that oligomycin did not affect mitochondrial repolarization in response to reperfusion. However, the simultaneous recording of cell length in Fig. 3B shows that no hypercontracture occurred on reperfusion with normal Tyrode in the presence of oligomycin. Overall, oligomycin reduced the proportion of hypercontracted cells to 6.6 ± 1.8% (n = 6; 70) compared to a control value of 83.4 ± 1.9% (p<0.01). These results suggest that, though mitochondrial repolarization is unaffected by oligomycin, the block of consequent ATP synthesis prevents hypercontracture.


Figure 3
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  		Fig. 3 Oligomycin prevents hypercontracture but not mitochondrial repolarization. (A) Recording of Mg Green fluorescence from a myocyte during MI and reperfusion. (B) Simultaneous recordings of TMRE fluorescence (relative to its initial value in normal Tyrode) and cell length from a myocyte during MI and reperfusion. Oligomycin (5 µg ml–1) was applied as indicated. (C) Mean values of relative TMRE fluorescence at the end of MI and after 10 min reperfusion in the absence (n = 5; 42) and presence of oligomycin as indicated, (n = 4; 32).

 
3.3. Loss of Ca2+ homeostasis and contractile function require Ca2+ influx during reperfusion but hypercontracture does not
To investigate the importance of Ca2+ influx during reperfusion on reperfusion-induced hypercontracture we reperfused cells with Ca2+-free Tyrode or inhibited reverse-mode Na+/Ca2+ exchange (NCX) with KB-R 7943. Fig. 4A shows a record of cell length from a single myocyte reperfused with Ca2+-free Tyrode after exposure to MI. The myocyte hypercontracted even in the absence of extracellular Ca2+. In similar experiments where 6–10 cells were observed simultaneously and exposed to MI Tyrode for 10 min and reperfused for 10 min, the absence of Ca2+ in the reperfusing solution did not reduce the proportion of myocytes that hypercontracted (Fig. 4B).


Figure 4
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  		Fig. 4 Calcium influx during reperfusion is not required for hypercontracture. (A) Recording of cell length from a single myocyte during MI and reperfusion with Ca2+-free Tyrode for 10 min. (B) Percentage of hypercontracted cells after 10 min reperfusion with normal Tyrode (control, n = 20; 158), or Ca2+-free Tyrode (0Ca, n = 34; 295).

 
In the model of cell injury following MI/reperfusion that we have used, reperfusion injury is characterized by the loss of Ca2+ homeostasis and contractile function as well as by hypercontracture. To investigate the role of Ca2+ entry during reperfusion, we have studied the effects on Ca2+ homeostasis and contractile function of reperfusing the myocytes in the absence of Ca2+ for 10 min prior to exposure to Ca2+-containing Tyrode. Fig. 5A shows intracellular Ca2+ recorded simultaneously from 10 myocytes. Superfusion with MI Tyrode resulted in contractile failure and a steady increase in [Ca2+]i to between 200 and 500 nM. Reperfusion with Ca2+-free Tyrode blocked the subsequent rise in [Ca2+]i, confirming that the increase in Ca2+ on reperfusion resulted from Ca2+ influx. In similar experiments we found that reperfusion with Tyrode containing 10 mM KB-R 7943 to inhibit NCX also prevented the rise in [Ca2+]i, consistent with Ca2+ entry occurring by reverse-mode NCX activity. When Ca2+ was readmitted following 10 min reperfusion with Ca2+-free Tyrode, [Ca2+]i rose sharply, but recovered to low levels in 5 out of 10 cells in the experiment of Fig. 5A. Similar effects were seen on the removal of KB-R 7943 (Fig. 5B). Fig. 5C and D show that the percentage of cells that recovered Ca2+ homeostasis (indicated by resting [Ca2+]i<250 nM after 10 min in normal Ca2+-containing Tyrode) was increased when Ca2+ influx was prevented during the first 10 min of reperfusion either by the absence of extracellular Ca2+ or by 10 µM KB-R 7943. The proportion of myocytes that recovered contraction in response to electrical stimulation was also increased by prevention of Ca2+ entry during initial reperfusion (Fig. 5D). Thus, although preventing Ca2+ influx at the time of reperfusion did not prevent hypercontracture (Fig. 4), it substantially increased both the percentage of cells that recovered Ca2+ homeostasis and contractile function.


Figure 5
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  		Fig. 5 Calcium influx during reperfusion contributes to loss of Ca2+ homeostasis and contractile function. (A) Simultaneous recordings of [Ca2+]i from ten myocytes within a single field of view during MI for 10 min and reperfusion with Ca2+-free Tyrode for 10 min and then normal Tyrode for 10 min. Each trace is the [Ca2+]i recorded from a different cell using an imaging system. (B) Recordings of [Ca2+]i showing the effect of reperfusion in the presence of KB-R 7943. (C) The percentage of myocytes which recover low diastolic [Ca2+]i (<250 nM) after reperfusion with 1) normal Tyrode for 20 min or 2) Ca2+-free Tyrode for 10 min and normal Tyrode for 10 min, or 3) normal Tyrode+10 µM KB-R 7943 for 10 min then normal Tyrode for 10 min, as illustrated in the protocols shown below. (Normal n = 43; 722, Ca2+-free Tyrode n = 8; 85 and KB-R 7943 n = 7; 91 and *p<0.01 vs. control). (D) Percentage of myocytes which recover contractile function (twitch in response to electrical stimulation), as per part (C). (Normal n = 20; 159, Ca2+-free Tyrode n = 7; 62 and KB-R 7943 n = 7; 59 and *p<0.01 vs. control).

 
3.4. Loss of Ca2+ homeostasis and contractile function result from mitochondrial permeability transition pore opening during reperfusion
On reperfusion after metabolic inhibition, mitochondrial repolarization and consequent Ca2+ accumulation are thought to favour the opening of MPTPs which may contribute to cell injury [3,10,18]. We therefore examined the effect of inhibiting MPTP opening with cyclosporin A [18] on the loss of Ca2+ homeostasis and contractile function.

Fig. 6A shows intracellular Ca2+ recorded simultaneously from 10 myocytes superfused with MI-Tyrode for 10 min. Reperfusion with normal Tyrode in the presence of 0.5 µM cyclosporin A resulted in a rapid fall in [Ca2+]i followed by a rapid hypercontracture in 9/10 cells (not shown) after which Ca2+ began to increase in 5 cells but remained low in the other 5. In addition, Ca2+ transients in response to electrical field stimulation are evident in the cells where Ca2+ remained low (the Ca2+ transients appear spread out in time due to the aliasing effect of the slow sampling rate, as we have described previously [14]). The Ca2+ transients ceased when electrical field stimulation was switched off, and indicate that the excitation–contraction coupling mechanisms had recovered in these cells, even though they had hypercontracted.


Figure 6
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  		Fig. 6 Cyclosporin A prevents loss of Ca2+ homeostasis and contractile function. (A) Simultaneous recordings of [Ca2+]i from ten myocytes within a single field of view during MI for 10 min and reperfusion with normal Tyrode+0.5 µM CsA for 10 min. The bar charts show: (B) percentage of cells hypercontracted, (n = normal 14; 110, + cyclosporine A 8; 85 and Ca2+-free+cyclosporine A 7; 91 and *p<0.01 vs. control) (C) percentage of cells with diastolic [Ca2+]i<250 nM (n = normal 43; 722, + cyclosporine A 12; 176 and Ca2+-free+cyclosporine A 6; 95) and (D) percentage of cells that recover contractile function (cell numbers as per B, *p<0.01 vs. control), after reperfusion with 1) normal Tyrode 20 min, 2) normal Tyrode+CsA 10 min then normal Tyrode 10 min and 3) Ca2+-free Tyrode+CsA 10 min then normal Tyrode for 10 min. The protocols are illustrated below.

 
Cyclosporin A (0.5 µM) did not affect the percentage of cells that were hypercontracted following 10 min of reperfusion with normal Tyrode (Fig. 6B). However, Fig. 6C and D show that the percentages of cells that were able to maintain low [Ca2+]i levels and that contracted in response to 1 Hz electrical stimulation were increased significantly in the presence of cyclosporin A. Preventing Ca2+ influx at the time of reperfusion in the presence of cyclosporin A should further inhibit opening of the MPTP. Consistent with this, reperfusion with Ca2+-free Tyrode was without effect on the percentage of cells that hypercontracted, but further increased the percentages of cells that recovered low [Ca2+]i levels and that recovered contractile function (Fig. 6B,C,D). Since cyclosporin A can inhibit calcineurin in addition to MPTP formation [19], we repeated some experiments with another MPTP inhibitor, sanglifehrin A, which does not have this additional effect [19]. In agreement with the effect of cyclosporin A, reperfusion following MI in the presence of sanglifehrin A increased the percentage of cells in which Ca2+ recovered to <250 nM to 71.0 ± 7.6% from a control value of 43.2 ± 3.3% (n = 5; 69 and 6; 77, respectively, p<0.01).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Appendix A. Data Supplement
 References
 
The injury of isolated ventricular myocytes by MI and reperfusion, characterized by irreversible hypercontracture, loss of Ca2+ homeostasis and contractile function [6,13,14], is similar to the response of intact heart tissue to ischaemia and reperfusion [12,20,21]. Here we have investigated the roles of mitochondrial re-energization and ATP synthesis, and Ca2+ influx in this process. We show that reperfusion-induced hypercontracture requires mitochondrial re-energization and increased ATP availability through oxidative phosphorylation, but does not require Ca2+ influx during reperfusion. However, prevention of Ca2+ influx or the presence of cyclosporin A or sanglifehrin A increases the subsequent ability of myocytes to recover Ca2+ homeostasis and contractile function. These findings suggest that the mitochondrial permeability transition pore (MPTP) opens in response to Ca2+ influx during reperfusion, initiating loss of Ca2+ homeostasis and contractile failure.

4.1. The importance of mitochondrial re-energization and ATP synthesis for hypercontracture
Reperfusion of ischaemic myocardium may result in a strong hypercontracture which in the intact myocardium is likely to cause severe cellular injury [1]. Indeed, inhibition of contraction with 2,3-butanedionemonaximine early during reperfusion reduces infarct size [22] and blocks reperfusion injury in anoxic cardiomyocytes [23,24]. Hypercontracture and contraction band formation have been proposed to result from increased availability of ATP combined with high [Ca2+]i [4,6].

Our data show that reperfusion after MI led to rapid mitochondrial repolarization, which was followed closely by a transient fall in [Ca2+]i and abrupt hypercontracture. They also support the requirement for ATP, since inhibiting the F0/F1 ATP synthetase with oligomycin at the time of reperfusion abolished hypercontracture (and ATP production). As the {Delta}{Psi}m is generated by the activity of proton pumps driven by electron transport and oxidation of the electron donors [NADH] and [FADH2], re-energization of the mitochondria and hypercontracture will depend upon the availability of NADH and FADH2. After MI, the removal of complex IV inhibition by cyanide during reperfusion should result in the rapid restoration of electron transport and repolarization of {Delta}{Psi}m driven by the high levels of [NADH] and [FADH2] present at the end of MI. The repolarized {Delta}{Psi}m drives ATP synthesis (see Fig. 7). We have shown previously that depleting cellular levels of [NADH] and [FADH2] by pre-treating myocytes with dinitrophenol decreases the incidence of hypercontracture during reperfusion [13]. Estimates of [ATP] using luciferin/luciferase suggest that a small rise in ATP is sufficient to trigger hypercontracture (100–200 µM) [4]. We found that reperfusion leads to a fall in Mg-Green fluorescence, indicating a partial recovery of ATP. These data are consistent with a critical role for increased ATP synthesis consequent on mitochondrial repolarization in hypercontracture and so reperfusion injury. However, Ca2+ influx during reperfusion does not appear to be required for the activation of hypercontracture, which occurs as [Ca2+]i is falling and also in the absence of extracellular Ca2+ (Fig. 2). In contrast, in reoxygenation-induced hypercontracture of cardiac myocytes Ca2+ influx during reperfusion contributes to large Ca2+ oscillations that precipitate a slowly-developing hypercontracture [7,25]. It is likely that this difference results from lower levels of [NADH] and [FADH2] at the end of hypoxia. Consistent with this, reperfusion-induced hypercontracture of ventricular myocytes following mitochondrial uncoupling with carbonylcyanide-m-chlorophenylhydrazone (CCCP), where [NADH] and [FADH2] are low, is slow, accompanied by large Ca2+ transients and dependent on extracellular Ca2+ [14].


Figure 7
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  		Fig. 7 Sequence of events leading to hypercontracture and loss of Ca2+ homeostasis and contractile function following reperfusion. Reperfusion re-energizes the mitochondria which through the restoration of electron transport causes repolarization of {Delta}{Psi}m. This rapid restoration of {Delta}{Psi}m leads to ATP-production by the F0/F1 synthetase which together with the high [Ca2+]i is responsible for the hypercontracture. Mitochondrial repolarization drives Ca2+ accumulation by the mitochondria, which together with production of ROS from the electron transport chain and the negative {Delta}{Psi}m triggers the formation and opening of MPTPs. Thus may lead to the eventual collapse of {Delta}{Psi}m and ATP-depletion via reverse F0/F1 synthetase. This decline in ATP through the inhibition of ATP-dependent Ca2+ pumps exacerbates the reperfusion-induced Ca2+ influx driven by the intracellular acidosis (Na+ accumulation via Na+/H+ exchange activity coupled to reverse-mode NCX).

 
4.2. Does Ca2+ influx during reperfusion trigger opening of the mitochondrial permeability transition pore?
Ca2+ influx through reverse-mode NCX at the time of reperfusion has been linked to the development of reoxygenation-induced hypercontracture in isolated cardiomyocytes, and loss of left ventricular function and release of LDH in ischaemic Langendorff-perfused hearts [7]. In our experiments, reperfusing the myocytes in the absence of extracellular Ca2+, or the presence of the NCX inhibitor KB-R 7943 [26,27], prevented the influx of Ca2+ but not hypercontracture. However, on subsequent re-admission of Ca2+ or removal of KB-R7943, the percentage of myocytes that recovered [Ca2+]i<250 nM and contracted in response to electrical stimulation was significantly increased. Cellular injury due to the Ca2+-paradox was prevented in these experiments by the presence of 1 mM MgCl2 in the Ca2+-free Tyrode [28]. These data suggest that, whilst mitochondrial re-energization and repolarization of {Delta}{Psi}m are the primary initiators of hypercontracture and loss of Ca2+ homeostasis and contractile function, the pathway leading to these different indicators of injury diverges soon afterwards (see Fig. 7).

Ischaemia/reperfusion injury in isolated hearts is characterized by depleted levels of high energy phosphates, in addition to ultrastructural damage resulting from hypercontracture [2,6]. Central to this depletion is the disruption of mitochondrial function which appears to result from the formation and opening of the MPTPs [3]. MPTP opening occurs during reperfusion and is initiated by mitochondrial repolarization, the production of free radicals and mitochondrial Ca2+ accumulation [3,10]. The presence of cyclosporin A or sanglifehrin A, which inhibit formation of MPTP, protected myocytes against reperfusion-induced loss of Ca2+ homeostasis and contractile function, and this effect was additive to that of Ca2+-free reperfusion (Fig. 6C,D). Cyclosporin A and sanglifehrin A did not, however, prevent hypercontracture. These findings suggest a central role for the MPTP in reperfusion-induced loss of Ca2+ homeostasis and contractile function. Opening of MPTPs causes irreversible depolarization of the mitochondria leading to rapid ATP depletion as the F0/F1 synthetase hydrolyses ATP [3], and production of ROS at complex I [29] (see Fig. 7). This inhibits cellular ATP-dependent processes, which contribute to loss of ionic homeostasis (Na+ and Ca2+) [3] and contractile dysfunction through inhibition of SERCA1 and ion channels (see Fig. 7). Paradoxically, using TMRE we did not see a sustained depolarization of {Delta}{Psi}m even after 10 min of reperfusion. This may have been due to the protective effect of TMRE itself, which liberates free radicals on excitation, leading to an increase in H2O2, which can reduce reperfusion injury [30,31]. This however, will not have affected the experiments we used to assess reperfusion-induced hypercontracture, loss of Ca2+ -homeostasis and contractile function as these cells were not loaded with TMRE.

This study suggests that the primary event which initiates the cascade responsible for reperfusion-injury following MI is mitochondrial re-energization, which repolarizes the mitochondria. It shows that increased availability of ATP but not Ca2+ influx during reperfusion initiates hypercontracture. During reperfusion the Ca2+ influx on reverse-mode NCX impairs functional recovery (contractile activity and ionic homeostasis), and this is probably linked to the MPTPs as the presence of cyclosporin A or sanglifehrin A enhances functional recovery. In the case of reperfusion of the intact ischaemic heart, reperfusion might be expected to induce a strong hypercontracture and mechanical injury at the centre of the ischaemic zone. However, at the margins of the zone, where partial perfusion from neighboring tissue may have occurred, hypercontracture may be less marked, but MPTP opening may still occur, compromising functional recovery.


   Appendix A. Data Supplement
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Data Supplement
References
 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2005.03.015.


   Acknowledgements
 
We thank Diane Everitt for expert technical assistance and the British Heart Foundation for support.


   Notes
 
Time for primary review 22 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Data Supplement
 References
 

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