AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 294: H386-H391, 2008. First published October 19, 2007; doi:10.1152/ajpheart.01035.2007
0363-6135/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/1/H386    most recent
01035.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Argaud, L.
Right arrow Articles by Ovize, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Argaud, L.
Right arrow Articles by Ovize, M.

Increased mitochondrial calcium coexists with decreased reperfusion injury in postconditioned (but not preconditioned) hearts

Laurent Argaud,1,2,* Odile Gateau-Roesch,1,* Lionel Augeul,1 Elisabeth Couture-Lepetit,1 Joseph Loufouat,1 Ludovic Gomez,1 Dominique Robert,2 and Michel Ovize1,2

1Institut National de la Santé et de la Recherche Médicale U886, Université Claude Bernard Lyon I, and 2Hospices Civils de Lyon, Lyon, France

Submitted 7 September 2007 ; accepted in final form 17 October 2007


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ca2+ is the main trigger for mitochondrial permeability transition pore opening, which plays a key role in cardiomyocyte death after ischemia-reperfusion. We investigated whether a reduced accumulation of mitochondrial Ca2+ might explain the attenuation of lethal reperfusion injury by postconditioning. Anesthetized New Zealand White rabbits underwent 30 min of ischemia, followed by either 240 (infarct size protocol) or 60 (mitochondria protocol) min of reperfusion. They received either no intervention (control), preconditioning by 5-min ischemia and 5-min reperfusion, postconditioning by four cycles of 1-min reperfusion and 1-min ischemia at the onset of reflow, or pharmacological inhibition of the transition pore opening by N-methyl-4-isoleucine-cyclosporin (NIM811; 5 mg/kg iv) given at reperfusion. Area at risk and infarct size were assessed by blue dye injection and triphenyltetrazolium chloride staining. Mitochondria were isolated from the risk region for measurement of 1) Ca2+ retention capacity (CRC), and 2) mitochondrial content of total (atomic absorption spectrometry) and ionized (potentiometric technique) calcium concentration. CRC averaged 0.73 ± 0.16 in control vs. 4.23 ± 0.17 µg Ca2+/mg proteins in shams (P < 0.05). Postconditioning, preconditioning, or NIM811 significantly increased CRC (P < 0.05 vs. control). In the control group, total and free mitochondrial calcium significantly increased to 2.39 ± 0.43 and 0.61 ± 0.10, respectively, vs. 1.42 ± 0.09 and 0.16 ± 0.01 µg Ca2+/mg in sham (P < 0.05). Surprisingly, whereas total and ionized mitochondrial Ca2+ decreased in preconditioning, it significantly increased in postconditioning and NIM811 groups. These data suggest that retention of calcium within mitochondria may explain the decreased reperfusion injury in postconditioned (but not preconditioned) hearts.

ischemia; preconditioning; mitochondria; myocardial infarction

ZHAO ET AL. RECENTLY REPORTED that repeated cycles of brief reperfusion and ischemia performed immediately at the onset of reperfusion following a prolonged ischemic insult dramatically limited infarct size. They named this phenomenon "postconditioning" (59). This cardioprotection, which is as powerful as ischemic preconditioning, has now been reported in several experimental preparations (9, 35, 56). Reports indicate that postconditioning may require activation of several kinases, including phosphatidylinositol 3-kinase, Akt, ERK1/2, PKC-{varepsilon}, and GSK-3β, as well as guanylyl-cyclase and endothelial nitric oxide synthase (8, 11, 19, 33, 49, 55, 58). Our laboratory recently demonstrated in the rabbit heart that ischemic postconditioning inhibits mitochondrial permeability transition pore (mPTP) opening, in keeping with data showing that cyclosporine A (CsA), a pharmacological inhibitor of mitochondrial permeability transition, reduces infarct size when administered at the time of reperfusion (3, 4, 28).

Mitochondrial permeability transition relates to the opening of a mega-channel, whose molecular structure is still debated. Opening of this large, nonspecific pore in the inner mitochondrial membrane results in the collapse of the membrane potential, matrix swelling, uncoupling of the respiratory chain, efflux of cytochrome c and other proapoptotic factors, and the F1-Fo ATPase hydrolyzing instead of building ATP (13). It is thought that mPTP opening is activated by binding of cyclophilin D (cypD) to the matrix side of a core protein; this binding is favored by intramitochondrial calcium accumulation (16, 25, 27). Griffiths and Halestrap (24) demonstrated that the mPTP remains closed throughout ischemia, but opens at the time of reperfusion. As a consequence of mPTP opening, a large amount of mitochondrial calcium is abruptly released into the cytosol, and calcium is recognized as a key player of reperfusion injury. Several reports demonstrated that mPTP opening plays a critical role in cardiomyocyte lethal reperfusion injury (17, 41, 53).

Postconditioning does attenuate lethal reperfusion injury. We hypothesized that ischemic postconditioning would be protective by preventing matrix calcium accumulation (and consequently inhibiting mPTP opening) following a prolonged ischemia-reperfusion. To address this hypothesis, we used quantitative techniques to measure total and ionized calcium accumulation, together with the calcium retention capacity (CRC), in cardiac mitochondria isolated from rabbit hearts that had been either postconditioned, preconditioned, or treated by N-methyl-4-isoleucine-cyclosporin (NIM811), a cypD inhibitor.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
The investigation conforms with the APS's Guiding Principles in the Care and Use of Animals and the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). All experimental procedures were approved by the Lyon I Claude Bernard University Committee for Animal Research.

Surgical preparation. Male New Zealand White rabbits, weighing 2.2 to 2.5 kg, were anesthetized by intramuscular injections of xylazine (5 mg/kg) and ketamine (50 mg/kg), as previously described (5). An intravenous infusion of a mixture of xylazine (20–50 µg·kg–1·min–1) and ketamine (40–100 µg·kg–1·min–1) was then maintained throughout the experiment. After a midline cervical incision, a tracheotomy was performed, and animals were ventilated with room air. A cannula was inserted into the right internal jugular vein for administration of drugs and fluids and into the left carotid artery for measurement of blood pressure. A left thoracotomy was performed in the fourth left intercostal space. The pericardium was opened, and the heart was exposed. A 3.0 silk suture attached to a small curved needle was passed around a marginal branch of the left circumflex coronary artery. Both ends of the thread were passed through a small vinyl tube to form a snare that could be tightened to occlude and loosened to reperfuse the artery. Body temperature was monitored via an intraperitoneal thermometer and kept constant by means of a heating pad. Hemodynamics (heart rate and arterial pressure) were monitored continuously throughout the experiment on a Gould recorder (Gould, Cleveland, OH). After the surgical procedure, a 20-min stabilization period was observed.

Experimental protocol. After the stabilization period, all animals underwent 30 min of coronary artery occlusion followed by either 60 min (isolated mitochondria protocol) or 240 min (infarct size protocol) of reperfusion (Fig. 1).


Figure 1
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 1. Experimental design. Sham underwent no ischemia-reperfusion. All animals underwent 30 min of ischemia followed by 240 min (infarct size study) or 60 min (mitochondria study) of reperfusion. Preconditioning (PreC) consisted of one episode of 5 min of ischemia and 5 min of reperfusion before the 30-min ischemia. Postconditioning (PostC) was induced by four cycles of 1-min reperfusion and 1-min occlusion at the onset of the final reperfusion period. N-methyl-4-isoleucine-cyclosporin (NIM811; a specific inhibitor of mitochondrial permeability transition pore opening) was administered as an intravenous bolus (arrows) 1 min before reperfusion.

 
In both protocols, control rabbits underwent no further intervention. Preconditioned animals (PreC group) received 5 min of ischemia followed by 5 min of reperfusion before the 30-min coronary artery occlusion. Postconditioning (PostC group) consisted in four cycles of 1 min of reperfusion followed by 1 min of ischemia, performed just after the prolonged ischemia. One group of rabbits received an intravenous bolus injection of 5 mg/kg of NIM811, a non-immunosuppressive derivative of CsA that specifically inhibits mPTP opening and prevents mitochondrial permeability transition (2, 51). This drug was administered 1 min before reperfusion, as a postconditioning mimetic (NIM811 group). Sham animals received no intervention for the whole duration of the experiment.

Measurement of area at risk and infarct size. At the end of the 4-h reperfusion, the coronary artery was briefly reoccluded, and 0.5 mg/kg Unisperse blue pigment (Ciba-Geigy, Hawthorne, NY) was injected intravenously to delineate the in vivo area at risk, as previously described (4). The heart was excised and cut into five to six 2-mm-thick transverse slices, parallel to the atrioventricular groove. Each slice was incubated for 15 min in a 1% solution of triphenyltetrazolium chloride at 37°C to differentiate infarcted (pale) from viable (brick red) myocardial area (50).

Preparation of isolated mitochondria. At the end of the experimental procedure, hearts were excised while still beating, immediately placed in a cold buffer A (70 mM sucrose, 210 mM mannitol, 1 mM EDTA in 50 mM Tris·HCl, pH 7.4), and area at risk myocardium were harvested for mitochondria isolation. For each heart, a single myocardial biopsy was used to assess CRC and both total and free mitochondrial Ca2+ content. Preparation of mitochondria was performed as previously described (2–4).

Mitochondrial oxygen consumption. Mitochondrial respiratory function was measured by the polarographic method of Chance and Williams using a Clark oxygen electrode (n = 4–5/group). The incubation media contained buffer B with 112 mM KCl, 6 mM MgCl2, and 10 mM NH2PO4, in 16 mM Tris·HCl/HEPES (pH 7.4, 25°C). Mitochondrial samples were added to incubation media containing FADH-dependent substrate succinate (2 mM). ADP (300 µM) was then added, and the following parameters were measured: state 3, oxygen consumption stimulated by ADP; and state 4, oxygen consumption after completion of ADP phosphorylation.

CRC. CRC represents a functional test for a quantitative assessment of the in vitro sensitivity of the mPTP to calcium load. CRC was assessed in sham (n = 12), control (n = 12), pre-C (n = 10), post-C (n = 9), and NIM811 (n = 9) groups. Adapted from the description by Ichas et al. (31), CRC was defined here as the amount of calcium required to trigger mPTP opening in isolated cardiac mitochondria that received a given in vivo intervention (e.g., postconditioning) (4). Briefly, isolated mitochondria (5 mg protein) were suspended in 100 µl buffer C (70 mM sucrose, 210 mM mannitol, 0.1 mM EDTA in 50 mM Tris·HCl, pH 7.4) and added in 900 µl of buffer D (150 mM sucrose, 50 mM KCl, 2 mM KH2PO4, 5 mM succinic acid in 20 mM Tris·HCl, pH 7.4) within a Teflon chamber equipped with a Ca2+-selective microelectrode, in conjunction with a reference electrode, as previously described (2, 3, 22, 30). Isolated mitochondria were gently stirred for 1 min. At the end of the preincubation period, 20 nmol CaCl2 pulses were performed every minute. In the mean time, modifications of the medium (i.e., extra-mitochondrial) Ca2+ concentration were continuously recorded using a custom-made Synchronie software. Following sufficient Ca2+ loading, extra-mitochondrial Ca2+ concentration abruptly increases, indicating a massive release of Ca2+ by mitochondria due to mPTP opening. CRC represents the amount of Ca2+ required to trigger this massive Ca2+ release; it is used here as an indicator of mPTP sensitivity to Ca2+ and expressed as micrograms of Ca2+ per milligram mitochondrial proteins. Following the isolation procedure, both the yield (ranging from 69 to 77%) and the respiratory control index were comparable among ischemia-reperfusion groups. In other words, any potential difference of CRC among groups cannot be due to a selection of specific subpopulation of mitochondria in any group during the isolation procedure.

Total mitochondrial Ca2+ content. Total mitochondrial Ca2+ content was assessed on pellets of 5-mg proteins after mineralization by HNO3 and using induced coupled plasma atomic emission spectrometry by means of a Maxim apparatus (Thermoptek). Measures were performed at the Service Central d'Analyse (CNRS, Vernaison, France). Results are expressed in micrograms Ca2+ per milligram mitochondrial proteins (n = 8–10/group).

Ionized mitochondrial Ca2+ content. Mitochondrial pellets (5 mg proteins) were resuspended in 100 µl 2% Triton X-100 in 20 mM Tris·HCl (pH 7.4) and freezed and thawed. The preparation was then added to 900 µl of buffer D within the Teflon chamber equipped with a Ca2+-selective microelectrode, as described earlier. Ionized Ca2+ was measured after calibration of the electrode using known amounts of Ca2+ to buffer C from a range of 0.005 to 0.5 mM CaCl2. Results are expressed in micrograms Ca2+ per milligram mitochondrial proteins (n = 8–10/group).

Chemicals. NIM811 used in the present study was a generous gift of Novartis (Basel, Switzerland). NIM811 was dissolved for in vivo use in a mixture of Cremophor EL (polyethoxylated castor oil) with 94% ethanol.

Statistical analysis. All values are expressed as means ± SE. Comparisons among groups were performed using one-way ANOVA. Means were compared by Fisher's test when a significant F value was obtained. Statistical significance was defined as a value of P < 0.05.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Infarct size. Thirty-one animals completed the infarct size protocol. Heart rate and blood pressure were comparable among groups throughout the protocol. All groups had a comparable size for area at risk. In the control group, infarct size averaged 59 ± 14% of the area at risk. Infarct size was significantly reduced to a similar extent in pre-C, post-C, as well as NIM811 groups, averaging 21 ± 7, 28 ± 9, and 28 ± 8% of the area at risk, respectively (Fig. 2).


Figure 2
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 2. Infarct size. Infarct size is expressed as percentage of the area at risk in four experimental groups: control (n = 8), Prec (n = 8), PostC (n = 7), and NIM811 (n = 8). Infarct size was significantly reduced in PreC, PostC, and NIM811 groups compared with controls. *P < 0.001 vs. control.

 
Mitochondrial respiration. After 60 min of reperfusion, state 3 had significantly decreased and state 4 significantly increased in control vs. sham mitochondria. Pre-C, post-C, and NIM811-treated groups displayed changes comparable to that observed in the control group (Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1. Mitochondrial respiration

 
CRC. In the sham group, the CRC averaged 4.23 ± 0.17 µg Ca2+/mg of mitochondrial proteins (Fig. 3). As expected, the CRC was significantly reduced in the control group, averaging 0.73 ± 0.16 µg Ca2+/mg proteins (P < 0.01 vs. sham). In the PostC, PreC, and NIM811 groups, the CRC was significantly improved compared with the control group (Fig. 3).


Figure 3
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 3. Calcium retention capacity (CRC). CRC was significantly reduced in control (n = 12) vs. sham group (n = 12). Both PreC (n = 10) and PostC (n = 9) equally attenuated the reduction of CRC resulting from prolonged ischemia-reperfusion. NIM811 (n = 9) further limited CRC decrease compared with control. *P < 0.05 vs. control; {dagger}P < 0.05 vs. PreC and PostC.

 
Total mitochondrial calcium content. In the sham group, total mitochondrial calcium content averaged 1.42 ± 0.09 µg Ca2+/mg proteins (Fig. 4). Following ischemia-reperfusion, total mitochondrial calcium content significantly increased up to 2.39 ± 0.43 µg Ca2+/mg proteins in the control group (P < 0.01 vs. sham). Importantly, compared with the control group, total mitochondrial calcium content was significantly reduced in the PreC but not in PostC group (Fig. 4). Total mitochondrial calcium content significantly increased in the NIM811-treated group (P < 0.01 vs. control) (Fig. 4).


Figure 4
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 4. Total and free mitochondrial calcium content. Total and free mitochondrial calcium content were significantly increased as a result of ischemia-reperfusion (n = 10), compared with the sham group (n = 10). PreC (n = 9), but not PostC (n = 9), significantly decreased both total and free mitochondrial calcium content. NIM811 treatment (n = 8) resulted in an even greater calcium accumulation. *P < 0.05 vs. sham; {dagger}P < 0.05 vs. control.

 
Ionized (free) mitochondrial Ca2+. Ischemia-reperfusion resulted in a significant increase in free mitochondrial Ca2+ that averaged 0.61 ± 0.10 µg Ca2+/mg proteins in the control group (P < 0.01 vs. 0.16 ± 0.01 µg Ca2+/mg proteins in the sham group) (Fig. 4). Free mitochondrial Ca2+ content was significantly reduced in PreC mitochondria, averaging 0.26 ± 0.05 µg Ca2+/mg proteins (P < 0.05 vs. control and PostC) (Fig. 4). In contrast, compared with the control group, postconditioning did not significantly modify free mitochondrial Ca2+ content that averaged 0.77 ± 0.15 µg Ca2+/mg proteins (P = not significant vs. control). Administration of NIM811 at the time of reperfusion increased mitochondrial content of free Ca2+ up to 2.58 ± 0.64 µg Ca2+/mg proteins (P < 0.05 vs. control) (Fig. 4).


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we demonstrated that mitochondria isolated from postconditioned rabbit hearts displayed an enhanced resistance to mitochondrial permeability transition, despite the absence of reduction of mitochondrial total and free calcium. These features were different from that seen in preconditioned hearts, but very similar to that observed in hearts that had been treated at reperfusion by the inhibitor of mPTP opening NIM811.

Mitochondrial calcium accumulation and lethal reperfusion injury. Using two different quantitative techniques, we found that total mitochondrial calcium content was significantly increased in control hearts after ischemia-reperfusion, along with a fourfold increase in matrix free Ca2+. Our results are in agreement with previous observations indicating that calcium accumulates within the cytosol and mitochondria during a prolonged ischemia-reperfusion (10, 18, 36, 45, 46, 52, 54). Evidence indicates that calcium accumulation plays a role in lethal reperfusion injury. Using indo 1 AM, Miyata et al. demonstrated in rat cardiomyocytes exposed to 35 min of anoxia followed by reoxygenation that both cytosolic and mitochondrial free Ca2+ concentrations rose after reoxygenation, and that the absolute magnitude of calcium increase predicts the recovery of cell function (39). Studies demonstrated that ruthenium red, which inhibits the mitochondrial calcium uniporter, reduces myocardial ischemia-reperfusion injury, likely by attenuating mitochondrial calcium uptake after reoxygenation, suggesting that mitochondrial but not cytosolic calcium was responsible for reperfusion injury (20, 38, 39). The mitochondrial uncoupler 2,4-dinitrophenol, administered at the time of reflow, attenuated the mitochondrial calcium gain and maintained cell membrane integrity, further suggesting a major role of mitochondrial calcium accumulation in lethal reperfusion injury (18).

Although mitochondrial calcium accumulation may play a role in lethal reperfusion injury, how it might trigger cell death remains unclear. Several studies support the idea that mitochondrial calcium accumulation would promote lethal reperfusion injury by activating mitochondrial permeability transition (15, 42). Our results are consistent with this paradigm. Compared with sham, control mitochondria that were loaded with calcium after ischemia-reperfusion displayed a reduced CRC, and control hearts developed large infarcts in this model (4).

To confirm this paradigm, one ought to determine whether limiting mitochondrial calcium accumulation can attenuate reperfusion injury in vivo. As a matter of fact, preconditioning significantly reduced total and free mitochondrial calcium, inhibited mitochondrial permeability transition, and reduced infarct size. The reduced calcium accumulation that we observed in preconditioned mitochondria is in concordance with previous reports from Steenbergen et al. (47) and others (1, 43, 52, 57), showing that preconditioning minimized ionic derangements during ischemia, such as an attenuation of the increase of intracellular calcium, and that this intervention was associated with improved recovery of contractility and less enzyme release on reflow. Reduced mitochondrial accumulation and limited triggering of mPTP opening would then likely be secondary to the reduction of the calcium gradient through the mitochondrial membrane. Further studies are, however, required to better understand how preconditioning might limit calcium accumulation within mitochondria. Our results in preconditioned hearts support the proposal for a crucial role of mitochondrial calcium accumulation in reperfusion injury.

Decreased reperfusion injury, despite calcium-loaded mitochondria in postconditioned hearts. We initially anticipated that postconditioning, which resembles preconditioning in several aspects, would also inhibit mitochondrial permeability transition and reduce infarct size via the limitation of mitochondrial calcium overload at reperfusion. Indeed, postconditioning reduces infarct size to a similar extent than preconditioning or the specific mPTP opening inhibitor NIM811 given at the time of reperfusion, underscoring the major role of mPTP opening in lethal reperfusion injury, as proposed by Lemasters’ group (29, 34) and Halestrap's group (24, 26, 32), as well as others (17). Furthermore, postconditioned mitochondria displayed an enhanced CRC, suggesting that postconditioning inhibits mPTP opening.

In our study, mitochondria were isolated (and CRC was assessed) after 60 min of reperfusion. An earlier time point was not chosen to avoid large variability in mitochondrial calcium concentration among experiments, partly due to increased instability in calcium homeostasis in the early minutes of reperfusion. Previous reports from our laboratory, as well as unpublished data, show that CRC changes are detectable in the early minutes following reflow and remain present at least for several hours thereafter (2, 4, 23).

One might question whether preconditioning, postconditioning, or NIM811 might have modified mitochondrial respiration during ischemia and reperfusion, and whether this might explain the observed changes in CRC. As expected, prolonged ischemia-reperfusion dramatically altered mitochondrial oxygen consumption with a decrease in state 3, suggesting an inhibition of the respiratory chain, and an increase in state 4, suggesting an uncoupling effect. But neither preconditioning nor postconditioning significantly altered mitochondrial oxygen consumption at 1 h of reperfusion, suggesting that the inhibition of mPTP opening in treated hearts was likely not directly related to a modification of mitochondrial respiration.

In sharp contrast with preconditioning, the beneficial effects of postconditioning occurred, despite the presence of a major accumulation of mitochondrial calcium. Both total and free calcium concentrations were as high in postconditioned as in control mitochondria. The larger accumulation of calcium in postconditioned vs. preconditioned mitochondria might simply be due to the fact that postconditioning that is triggered after the prolonged ischemia cannot limit calcium accumulation during this period. However, it is unlikely to explain such a large difference, since most of the calcium accumulation within mitochondria after a prolonged ischemic insult occurs at the time of reflow (36, 45, 46, 54). Our results are in apparent contradiction with those of Sun et al. (48), who first reported, using primary cultured neonatal rat cardiomyocytes, that hypoxic postconditioning was associated with a decrease in intracellular and mitochondrial calcium concentrations. This discrepancy is unclear, but might be related to important differences in experimental models, in the techniques used for measurement of ionized calcium, and to the fact that Sun et al. did not assess total mitochondrial calcium content.

While preconditioning protected the heart likely through a limitation of mitochondrial calcium accumulation, the coexistence of a massive calcium accumulation, together with an enhanced CRC, led us to propose that postconditioning might have desensitized the mPTP to calcium. This proposal is supported by the results obtained with NIM811. In comparable experimental conditions, NIM811 inhibited mitochondrial permeability transition via its action on cypD and reduced infarct size, despite a major increase in the accumulation of calcium within the mitochondrial matrix (51). cypD belongs to the family of peptidyl prolyl cis-trans isomerases that can bind CsA and its analogs, including NIM811 (12, 21). Although its specific role is unclear, this mitochondrial-targeted peptidyl prolyl cis-trans isomerase has been shown to play a key role in mitochondrial permeability transition, where the calcium requirement for the induction of the mPTP opening might be due to the calcium-dependent interaction between cypD and a pore protein (14, 15, 37, 44). The central role of cypD in lethal ischemia-reperfusion injury was recently confirmed by reports indicating that cardiac mitochondria isolated from cypD-deficient mice are resistant to calcium loading-induced mitochondrial permeability transition, and that these transgenic mice, as well as CsA-pretreated wild-type mice, develop smaller infarcts following a prolonged ischemia-reperfusion (6, 7, 40). Since postconditioning and NIM811 had very similar effects on infarct size, CRC, and accumulation of calcium within mitochondria, our results are consistent with the idea that postconditioning was protective by preventing the release of calcium by the mitochondrial transition pore at the time of reperfusion. How brief episodes of ischemia and reperfusion performed at the onset of reperfusion following a prolonged ischemic insult may, directly or indirectly, alter calcium sensitivity of the mPTP remains, however, to be determined.


   ACKNOWLEDGMENTS
 
We thank Dr. R. A. Kloner for overall discussion and Dr. E. Fontaine for discussion on mitochondrial permeability transition.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. Argaud, Inserm U866-Laboratoire de Physiologie Lyon-Nord, 8, Ave. Rockefeller, 69373 Lyon Cedex, France (e-mail: laurent.argaud{at}chu-lyon.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* L. Argaud and O. Gateau-Roesch contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. An J, Varadarajan SG, Novalija E, Stowe DF. Ischemic and anesthetic preconditioning reduces cytosolic [Ca2+] and improves Ca2+ responses in intact hearts. Am J Physiol Heart Circ Physiol 281: H1508–H1523, 2001.[Abstract/Free Full Text]
  2. Argaud L, Gateau-Roesch O, Chalabreysse L, Gomez L, Loufouat J, Thivolet-Béjui F, Robert D, Ovize M. Preconditioning delays Ca2+-induced mitochondrial permeability transition. Cardiovasc Res 61: 115–122, 2004.[Abstract/Free Full Text]
  3. Argaud L, Gateau-Roesch O, Muntean D, Chalabreysse L, Loufouat J, Robert D, Ovize M. Specific inhibition of the mitochondrial permeability transition prevents lethal reperfusion injury. J Mol Cell Cardiol 38: 367–374, 2005.[CrossRef][Medline]
  4. Argaud L, Gateau-Roesch O, Raisky O, Loufouat J, Robert D, Ovize M. Postconditioning inhibits mitochondrial permeability transition. Circulation 111: 194–197, 2005.[Abstract/Free Full Text]
  5. Argaud L, Prigent AF, Chalabreysse L, Loufouat J, Lagarde M, Ovize M. Ceramide in the antiapoptotic effect of ischemic preconditioning. Am J Physiol Heart Circ Physiol 286: H246–H251, 2004.[Abstract/Free Full Text]
  6. Baines CP, Kaiser RA, Purcell NH, Blair NS, Osinska H, Hambleton MA, Brunskill EW, Sayen MR, Gottlieb RA, Dorn GW, Robbins J, Molkentin JD. Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature 434: 658–662, 2005.[CrossRef][Medline]
  7. Basso E, Fante L, Fowlkes J, Petronilli V, Forte MA, Bernardi P. Properties of the permeability transition pore in mitochondria devoid of cyclophilin D. J Biol Chem 280: 18558–18561, 2005.[Abstract/Free Full Text]
  8. Bopassa JC, Ferrera R, Gateau-Roesch O, Couture-Lepetit E, Ovize M. PI3-kinase regulates the mitochondrial transition pore in controlled reperfusion and postconditioning. Cardiovasc Res 69: 178–185, 2006.[Abstract/Free Full Text]
  9. Bopassa JC, Michel P, Gateau-Roesch O, Ovize M, Ferrera R. Low-pressure reperfusion alters mitochondrial permeability transition. Am J Physiol Heart Circ Physiol 288: H2750–H2755, 2005.[Abstract/Free Full Text]
  10. Brierley GP, Murer E, Bachmann E. Studies on ion transport. III. The accumulation of calcium and inorganic phosphate by heart mitochondria. Arch Biochem Biophys 105: 89–102, 1964.[CrossRef][Web of Science][Medline]
  11. Chiari PC, Bienengraeber MW, Pagel PS, Krolikowski JG, Kersten JR, Warltier DC. Isoflurane protects against myocardial infarction during early reperfusion by activation of phosphatidylinositol-3-kinase signal transduction: evidence for anesthetic-induced postconditioning in rabbits. Anesthesiology 102: 102–109, 2005.[CrossRef][Web of Science][Medline]
  12. Connern CP, Halestrap AP. Purification and N-terminal sequencing of peptidyl-prolyl cis-trans-isomerase from rat liver mitochondrial matrix reveals the existence of a distinct mitochondrial cyclophilin. Biochem J 284: 381–385, 1992.[Web of Science][Medline]
  13. Crompton M. Mitochondrial intermembrane junctional complexes and their role in cell death. J Physiol 529: 11–21, 2000.[Abstract/Free Full Text]
  14. Crompton M. On the involvement of mitochondrial intermembrane junctional complexes in apoptosis. Curr Med Chem 10: 1473–1474, 2003.[CrossRef][Web of Science][Medline]
  15. Crompton M. The mitochondrial permeability transition pore and its role in cell death. Biochem J 341: 233–249, 1999.[CrossRef][Web of Science][Medline]
  16. Crompton M, Ellinger H, Costi A. Inhibition by cyclosporin A of a Ca2+-dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress. Biochem J 255: 357–360, 1988.[Web of Science][Medline]
  17. Di Lisa F, Menabo R, Canton M, Barile M, Bernardi P. Opening of the mitochondrial permeability transition pore causes depletion of mitochondrial and cytosolic NAD+ and is a causative event in the death of myocytes in postischemic reperfusion of the heart. J Biol Chem 276: 2571–2575, 2001.[Abstract/Free Full Text]
  18. Elz JS, Nayler WG. Calcium gain during postischemic reperfusion. The effect of 2,4-dinitrophenol. Am J Pathol 131: 137–145, 1988.[Abstract]
  19. Feng J, Lucchinetti E, Ahuja P, Pasch T, Perriard JC, Zaugg M. Isoflurane postconditioning prevents opening of the mitochondrial permeability transition pore through inhibition of glycogen synthase kinase-3β. Anesthesiology 103: 987–995, 2005.[CrossRef][Web of Science][Medline]
  20. Ferrari R, Di Lisa F, Raddino R, Visioli O. The effects of ruthenium red on mitochondrial function during post-ischaemic reperfusion. J Mol Cell Cardiol 14: 737–740, 1982.[CrossRef][Web of Science][Medline]
  21. Fischer G, Wittmann-Liebold B, Lang K, Kiefhaber T, Schmid FX. Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins. Nature 337: 476–478, 1989.[CrossRef][Medline]
  22. Fiskum G, Kowaltowski AJ, Andreyev AY, Kushnareva YE, Starkov AA. Apoptosis-related activities measured with isolated mitochondria and digitonin-permeabilized cells. Methods Enzymol 322: 222–234, 2000.[Web of Science][Medline]
  23. Gomez L, Chavanis N, Argaud L, Chalabreysse L, Gateau-Roesch O, Ninet J, Ovize M. Fas-independent mitochondrial damage triggers cardiomyocyte death after ischemia-reperfusion. Am J Physiol Heart Circ Physiol 289: H2153–H2158, 2005.[Abstract/Free Full Text]
  24. Griffiths EJ, Halestrap AP. Mitochondrial non-specific pores remain closed during cardiac ischemia but open upon reperfusion. Biochem J 307: 93–98, 1995.[Web of Science][Medline]
  25. Gunter TE, Pfeiffer DR. Mechanisms by which mitochondria transport calcium. Am J Physiol Cell Physiol 258: C755–C786, 1990.[Abstract/Free Full Text]
  26. Halestrap AP, Clarke SJ, Javadov SA. Mitochondrial permeability transition pore opening during myocardial reperfusion. A target for cardioprotection. Cardiovasc Res 61: 372–385, 2004.[Abstract/Free Full Text]
  27. Halestrap AP, Davidson AM. Inhibition of Ca2+-induced large-amplitude swelling of liver and heart mitochondria by cyclosporin is probably caused by the inhibitor binding to mitochondrial-matrix peptidyl-prolyl cis-trans isomerase and preventing it interacting with the adenine nucleotide translocase. Biochem J 268: 153–160, 1990.[Web of Science][Medline]
  28. Hausenloy DJ, Maddock HL, Baxter GF, Yellon DM. Inhibiting mitochondrial permeability transition pore opening: a new paradigm for myocardial preconditioning? Cardiovasc Res 55: 534–543, 2002.[Abstract/Free Full Text]
  29. He L, Lemasters JJ. Regulated and unregulated mitochondrial permeability transition pores: a new paradigm of pore structure and function? FEBS Lett 512: 1–7, 2002.[CrossRef][Web of Science][Medline]
  30. Holmuhamedov E, Jovanovic S, Dzeja P, Jovanovic A, Terzic A. Mitochondrial ATP-sensitive K+ channels modulate cardiac mitochondrial function. Am J Physiol Heart Circ Physiol 275: H1567–H1576, 1998.[Abstract/Free Full Text]
  31. Ichas F, Jouaville LS, Sidasha SS, Mazat JP, Holmuhamedov EL. Mitochondrial calcium spiking: a transduction mechanism based on calcium-induced permeability transition involved in cell calcium signalling. FEBS Lett 348: 211–215, 1994.[CrossRef][Web of Science][Medline]
  32. Javadov SA, Clarke S, Das M, Griffiths EJ, Lim KH, Halestrap AP. Ischaemic preconditioning inhibits opening of mitochondrial permeability transition pores in the reperfused rat heart. J Physiol 549: 513–524, 2003.[Abstract/Free Full Text]
  33. Jiang R, Maynard M, Whittaker P, Vinten-Johansen J, Przyklenk K. Postconditioning via stuttering reperfusion limits myocardial infarct size in rabbit hearts: role of ERK1/2. Am J Physiol Heart Circ Physiol 289: H1618–H1626, 2005.[Abstract/Free Full Text]
  34. Kim JS, He L, Lemasters JJ. Mitochondrial permeability transition: a common pathway to necrosis and apoptosis. Biochem Biophys Res Commun 304: 463–470, 2003.[CrossRef][Web of Science][Medline]
  35. Kin H, Zhao ZQ, Sun HY, Wang NP, Corvera JS, Halkos ME, Kerendi F, Guyton RA, Vinten-Johansen J. Postconditioning attenuates myocardial ischemia-reperfusion injury by inhibiting events in the early minutes of reperfusion. Cardiovasc Res 62: 74–85, 2004.[Abstract/Free Full Text]
  36. Kloner RA, Ganote CE, Whalen DA Jr, Jennings RB. Effect of a transient period of ischemia on myocardial cells. Am J Pathol 74: 399–422, 1974.[Web of Science][Medline]
  37. Lin DT, Lechleiter JD. Mitochondrial targeted cyclophilin D protects cells from cell death by peptidyl prolyl isomerization. J Biol Chem 277: 31134–31141, 2002.[Abstract/Free Full Text]
  38. Miyamae M, Camacho SA, Weiner MW, Figueredo VM. Attenuation of postischemic reperfusion injury is related to prevention of [Ca2+]m overload in rat hearts. Am J Physiol Heart Circ Physiol 271: H2145–H2153, 1996.[Abstract/Free Full Text]
  39. Miyata H, Lakatta EG, Stern MD, Silverman HS. Relation of mitochondrial and cytosolic free calcium to cardiac myocyte recovery after exposure to anoxia. Circ Res 71: 605–613, 1992.[Abstract/Free Full Text]
  40. Nakagawa T, Shimizu S, Watanabe T, Yamaguchi Otsu K, Yamagata H, Inohara H, Kubo T, Tsujimoto Y. Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death. Nature 434: 652–657, 2005.[CrossRef][Medline]
  41. Nazareth W, Yafei N, Crompton M. Inhibition of anoxia-induced injury in heart myocytes by cyclosporin A. J Mol Cell Cardiol 23: 1351–1354, 1991.[CrossRef][Web of Science][Medline]
  42. Rizzuto R, Bernardi P, Pozzan T. Mitochondria as all-round players of the calcium game. J Physiol 529: 37–47, 2000.[Abstract/Free Full Text]
  43. Saini HK, Dhalla NS. Defective calcium handling in cardiomyocytes isolated from hearts subjected to ischemia-reperfusion. Am J Physiol Heart Circ Physiol 288: H2260–H2270, 2005.[Abstract/Free Full Text]
  44. Schubert A, Grimm S. Cyclophilin D, a component of the permeability transition-pore, is an apoptosis repressor. Cancer Res 64: 85–93, 2004.[Abstract/Free Full Text]
  45. Shen AC, Jennings RB. Myocardial calcium and magnesium in acute ischemic injury. Am J Pathol 67: 417–40, 1972.[Web of Science][Medline]
  46. Shen AC, Jennings RB. Kinetics of calcium accumulation in acute myocardial ischemic injury. Am J Pathol 67: 441–452, 1972.[Web of Science][Medline]
  47. Steenbergen C, Perlman ME, London RE, Murphy E. Mechanism of preconditioning ionic alterations. Circ Res 72: 112–115, 1993.[Abstract/Free Full Text]
  48. Sun HY, Wang NP, Kerendi F, Halkos M, Kin H, Guyton RA, Vinten-Johansen J, Zhao ZQ. Hypoxic postconditioning reduces cardiomyocyte loss by inhibiting ROS generation and intracellular Ca2+ overload. Am J Physiol Heart Circ Physiol 288: H1900–H1908, 2005.[Abstract/Free Full Text]
  49. Tsang A, Hausenloy DJ, Mocanu MM, Yellon DM. Postconditioning: a form of "modified reperfusion" protects the myocardium by activating the phosphatidylinositol 3-kinase-Akt pathway. Circ Res 95: 230–232, 2004.[Abstract/Free Full Text]
  50. Vivaldi MT, Kloner RA, Schoen FJ. Triphenyltetrazolium staining of irreversible ischemic injury following coronary artery occlusion in rats. Am J Pathol 121: 522–530, 1985.[Abstract]
  51. Waldmeier PC, Feldtrauer JJ, Qian T, Lemasters JJ. Inhibition of the mitochondrial permeability transition by the nonimmunosuppressive cyclosporin derivative NIM811. Mol Pharmacol 62: 22–29, 2002.[Abstract/Free Full Text]
  52. Wang L, Cherednichenko G, Hernandez L, Halow J, Camacho A, Figueredo V, Schaefer S. Preconditioning limits mitochondrial Ca2+ during ischemia in rat hearts: role of KATP channels. Am J Physiol Heart Circ Physiol 280: H2321–H2328, 2001.[Abstract/Free Full Text]
  53. Weiss JN, Korge P, Honda HM, Ping P. Role of the mitochondrial permeability transition in myocardial disease. Circ Res 93: 292–301, 2003.[Abstract/Free Full Text]
  54. Whalen DA Jr, Hamilton DG, Ganote CE, Jennings RB. Effect of a transient period of ischemia on myocardial cells. I. Effects on cell volume regulation. Am J Pathol 74: 381–397, 1974.[Web of Science][Medline]
  55. Yang XM, Philipp S, Downey JM, Cohen MV. Postconditioning's protection is not dependent on circulating blood factors or cells but involves adenosine receptors and requires PI3-kinase and guanylyl cyclase activation. Basic Res Cardiol 100: 57–63, 2005.[CrossRef][Web of Science][Medline]
  56. Yang XM, Proctor JB, Cui L, Krieg T, Downey JM, Cohen MV. Multiple, brief coronary occlusions during early reperfusion protect rabbit hearts by targeting cell signaling pathways. J Am Coll Cardiol 44: 1103–1110, 2004.[Abstract/Free Full Text]
  57. Ylitalo KV, Ala-Rami A, Liimatta EV, Peuhkurinen KJ, Hassinen IE. Intracellular free calcium and mitochondrial membrane potential in ischemia/reperfusion and preconditioning. J Mol Cell Cardiol 32: 1223–1238, 2000.[CrossRef][Web of Science][Medline]
  58. Zatta AJ, Kin H, Lee G, Wang N, Jiang R, Lust R, Reeves JG, Mykytenko J, Guyton RA, Zhao ZQ, Vinten-Johansen J. Infarct-sparing effect of myocardial postconditioning is dependent on protein kinase C signalling. Cardiovasc Res 70: 315–324, 2006.[Abstract/Free Full Text]
  59. Zhao ZQ, Corvera JS, Halkos ME, Kerendi F, Wang NP, Guyton RA, Vinten-Johansen J. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol 285: H579–H588, 2003.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
A. Granfeldt, D. J. Lefer, and J. Vinten-Johansen
Protective ischaemia in patients: preconditioning and postconditioning
Cardiovasc Res, July 15, 2009; 83(2): 234 - 246.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/1/H386    most recent
01035.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Argaud, L.
Right arrow Articles by Ovize, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Argaud, L.
Right arrow Articles by Ovize, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.