AJP - Heart Journal of Neurophysiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 293: H1654-H1661, 2007. First published June 8, 2007; doi:10.1152/ajpheart.01378.2006
0363-6135/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/3/H1654    most recent
01378.2006v1
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 (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gomez, L.
Right arrow Articles by Ovize, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gomez, L.
Right arrow Articles by Ovize, M.

Inhibition of mitochondrial permeability transition improves functional recovery and reduces mortality following acute myocardial infarction in mice

Ludovic Gomez,1 Hélène Thibault,1,2 Adbdallah Gharib,1 Jean-Maurice Dumont,3 Grégoire Vuagniaux,3 Pietro Scalfaro,3 Geneviève Derumeaux,1,2 and Michel Ovize1,2

1Institut National de la Santé et de la Recherche Médicale E 0226, Université Claude Bernard Lyon I, Lyon, and 2Hôpital Louis Pradel, Hospices Civils de Lyon, Lyon Cedex, France; and 3DebioPharm S.A., Lausanne, Switzerland

Submitted 18 December 2006 ; accepted in final form 1 June 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhibition of mitochondrial permeability transition pore (mPTP) opening by cyclosporin A or ischemic postconditioning attenuates lethal reperfusion injury. Its impact on major post-myocardial infarction events, including worsening of left ventricular (LV) function and death, remains unknown. We sought to determine whether pharmacological or postconditioning-induced inhibition of mPTP opening might improve functional recovery and survival following myocardial infarction in mice. Anesthetized mice underwent 25 min of ischemia and 24 h (protocol 1) or 30 days (protocol 2) of reperfusion. At reperfusion, they received no intervention (control), postconditioning (3 cycles of 1 min ischemia-1 min reperfusion), or intravenous injection of the mPTP inhibitor Debio-025 (10 mg/kg). At 24 h of reperfusion, mitochondria were isolated from the region at risk for assessment of the Ca2+ retention capacity (CRC). Infarct size was measured by triphenyltetrazolium chloride staining. At 30 days of reperfusion, mortality and LV contractile function (echocardiography) were evaluated. Postconditioning and Debio-025 significantly improved Ca2+ retention capacity (132 ± 13 and 153 ± 31 vs. 53 ± 16 nmol Ca2+/mg protein in control) and reduced infarct size to 35 ± 4 and 32 ± 7% of area at risk vs. 61 ± 6% in control (P < 0.05). At 30 days, ejection fraction averaged 74 ± 6 and 77 ± 6% in postconditioned and Debio-025 groups, respectively, vs. 62 ± 12% in the control group (P < 0.05). At 30 days, survival was improved from 58% in the control group to 92 and 89% in postconditioned and Debio-025 groups, respectively. Inhibition of mitochondrial permeability transition at reperfusion improves functional recovery and mortality in mice.

ischemic postconditioning; left ventricular contractile function; mitochondrial permeability transition pore

MYOCARDIAL INFARCTION (MI) remains the leading cause of cardiac death in Western countries. Heart failure is an increasingly common outcome of MI and a frequent cause of cardiovascular morbidity and mortality (22, 40, 44). Survival 5 yr after the diagnosis of heart failure is poor, as low as 25–35% (33).

Infarct size is a major determinant of mortality (10). Besides treatment of heart failure per se, initial limitation of infarct size appears, in theory, to be the best strategy to prevent postischemic heart failure and improve survival.

Postconditioning might offer major hope to improve postinfarction outcome. Zhao et al. (54) reported that three episodes of 30 s of reperfusion-30 s of ischemia immediately after a prolonged 60-min ischemic insult in the dog heart dramatically attenuate irreversible myocardial injury. This observation has been confirmed in several experimental preparations and, more recently, in humans (4, 9, 32, 35, 38, 47).

Since all patients with ongoing acute MI cannot be treated with ischemic postconditioning by angioplasty, identification of a pharmacological mimetic to postconditioning is obviously required. Potential molecular targets have been identified, along with signaling pathways involved in postconditioning, including adenosine A2A or A3 receptors, mitochondrial ATP-sensitive K+ channels, and several kinases, including phosphatidylinositol 3-kinase, Akt, endothelial nitric oxide synthase, PKC-{epsilon}, ERK1/2, and glycogen synthase kinase-3beta (11, 14, 17, 37, 48, 52, 53). These signal transduction pathways seem to converge to mitochondria and, more specifically, implicate a function called "mitochondrial permeability transition" (12, 15, 50, 55). Convincing evidence indicates that mitochondrial permeability transition plays a crucial role in lethal myocardial reperfusion injury (16, 27, 28, 31, 36). Argaud et al. (4, 5) demonstrated in the in vivo rabbit heart that ischemic postconditioning inhibits mitochondrial permeability transition pore (mPTP) opening and pharmacological inhibition of mPTP opening by cyclosporin A (CsA) and its analog NIM-811, administered at the onset of reperfusion, reduced infarct size to an extent similar to that caused by ischemic postconditioning.

Although postconditioning by ischemia or mPTP inhibition can reduce infarct size, it remains unknown whether both interventions provide a persistent beneficial outcome following acute MI. Using the mouse model of MI, with a 30-day follow-up period, we questioned whether inhibition of mPTP opening at the time of reperfusion, by ischemic postconditioning or a pharmacological intervention (i.e., 2 realistic clinical interventions), would enhance recovery of contractile function and improve survival.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996) and performed under a license (No. 69.388.0502) from the French government (veterinary department) to conduct animal research.

Surgical Preparation

Seven- to 8-wk-old male OF1 mice (Charles River Laboratories, L'Arbresle, France) were anesthetized by intraperitoneal injection of 0.3 ml/10 g body wt of a 1:1 mixture of fentanyl citrate (0.011 mg/ml) and midazolam (0.4 mg/ml), as previously described (23). The animals were orally intubated using a 22-gauge vinyl catheter and ventilated via a rodent ventilator (model 687, Harvard Apparatus), with a tidal volume of 0.2 ml and a breath rate of 160 beats/min. A left thoracotomy was performed in the fourth left intercostal space. The pericardium was opened, and the heart was exposed. An 8-0 polypropylene suture attached to a small curved needle was passed around the left anterior descending coronary artery. Ischemia was confirmed by ST segment shift on the ECG (78534C monitor, Hewlett Packard) and the appearance of myocardial pallor. Body temperature was monitored via a rectal thermometer and maintained at 36–37°C with a heating pad. After surgery, the animals were allowed to recover from anesthesia, and the endotracheal tube was removed once spontaneous breathing resumed.

Experimental Design

All animals underwent 25 min of coronary artery occlusion followed by reperfusion (Fig. 1). Postconditioning consisted of three cycles of 1 min of reperfusion and 1 min of ischemia performed immediately after reflow. In the Debio-025 group, 5 min before reperfusion, mice received a slow (over 3 min) injection of Debio-025 (10 mg/kg iv). Control mice received the vehicle for Debio-025 (a mixture of Solutol and 94% ethanol) (46) under similar conditions. At the end of the surgical procedure, the chest was closed, and the animal was returned to the animal facility until the end of the reperfusion period.


Figure 1
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 1. Experimental design. All groups underwent 25 min of ischemia followed by 24 h (infarct size and isolated mitochondria experiments) or 30 days (assessment of contractile function and survival) of reperfusion. Debio-025 was injected intravenously 5 min before reperfusion. Postconditioning (PostC) consisted of 3 episodes of 1 min of reperfusion followed by 1 min of ischemia (3 x 1). LV, left ventricle.

 
Protocol 1: infarct size and mitochondrial permeability transition. After 24 h of reperfusion, a first subset of mice were euthanized under deep anesthesia, and the heart was excised for determination of infarct size (n = 28) or assessment of Ca2+-induced mitochondrial permeability transition (n = 21).

Protocol 2: recovery of LV function and survival. An additional subset of mice (n = 57) underwent 30 days of reperfusion. During the 30-day study period, the cages were inspected daily to identify any deceased animal. At the end of the 30-day period (i.e., just before euthanasia), echocardiography was performed under light intraperitoneal anesthesia.

Techniques

Area at risk and infarct size determination. At the end of the 24-h reperfusion, the coronary artery was briefly reoccluded, and Unisperse blue pigment (0.5 mg/kg iv; Ciba-Geigy, Hawthorne, NY) was injected to delineate the in vivo area at risk, as previously described (23). With this technique, the previously nonischemic myocardium appears blue, whereas the previously ischemic myocardium (area at risk) remains unstained.

The heart was excised and cut into four to five 1-mm-thick transverse slices, parallel to the atrioventricular groove. After removal of right ventricular tissue, each heart slice was weighed. The basal surface of each slice was photographed for later measurement of the area at risk. Each slice was then incubated for 15 min in a 1% solution of triphenyltetrazolium chloride at 34°C to differentiate infarcted (pale) from viable (brick red) myocardial area (49). The slices were then rephotographed. Enlarged projections of these slices were traced for determination of the boundaries of the area at risk and area of necrosis. Extent of the area at risk and area of necrosis was quantified by computerized planimetry and corrected for the weight of the tissue slices.

Total weights of the area at risk and the area of necrosis were then calculated and expressed in grams and as percentage of total LV or area at risk weight, respectively.

Preparation of isolated mitochondria. At the end of the 24-h reperfusion period, hearts were excised while still beating and immediately placed in cold buffer, and area at risk myocardium was harvested for mitochondria isolation. Preparation of mitochondria was adapted from a previously described procedure (23). All operations were carried out in a cold room at 4°C. Myocardial area at risk biopsies (20–30 mg) were placed in isolation buffer containing 70 mM sucrose, 210 mM mannitol, and 1 mM EDTA in 50 mM Tris·HCl (pH 7.4). The tissue was finely minced with scissors and then homogenized in the same buffer (10 µl buffer/mg tissue) with a Kontes tissue grinder and then with a Potter-Elvehjem. The homogenate was centrifuged at 1,300 g for 3 min and the supernatant at 10,000 g for 10 min. The mitochondrial pellet was suspended in isolation buffer containing 70 mM sucrose, 210 mM mannitol, and 0.1 mM EDTA in 50 mM Tris·HCl (pH 7.4). Protein content was routinely assayed according to the procedure of Gornall et al. (25), with bovine serum albumin used as the standard.

Ca2+ retention capacity. Extramitochondrial Ca2+ concentration was measured with a Hitachi F2500 spectrofluorometer in the presence of 0.5 µM Calcium Green-5N, with excitation and emission wavelengths set at 500 and 530 nm, respectively. Briefly, isolated mitochondria (100 µg of protein) were suspended in 2 ml of buffer [150 mM sucrose, 50 mM KCl, 2 mM KH2PO4, and 5 mM succinic acid in 20 mM Tris·HCl (pH 7.4)] in a polystyrene cuvette. Mitochondria were gently stirred for 90 s. At the end of the preincubation period, 5-nmol CaCl2 pulses were administered every 60 s. Each 5-nmol CaCl2 injection causes a peak of extramitochondrial Ca2+ concentration; then extramitochondrial Ca2+ concentration spontaneously returns to near-baseline level as Ca2+ enters the mitochondrial matrix via the Ca2+ uniporter (7) (see GoFig. 3A). After sufficient Ca2+ loading, extramitochondrial Ca2+ concentration abruptly increased, indicating a massive release of Ca2+ by mitochondria due to mPTP opening, as previously described (23, 24). Ca2+ retention capacity (CRC) was defined as the amount of Ca2+ required to trigger this massive Ca2+ release (3, 34); it is used here as an indicator of the mPTP sensitivity to Ca2+ and expressed as nanomoles of CaCl2 per milligram of mitochondrial proteins (n = 5–6/group).


Figure 2
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 2. In vitro effects of Debio-025, measured as Ca2+ retention capacity (CRC), on isolated cardiac mitochondria. Dose-dependent inhibition of mitochondrial permeability transition pore (mPTP) opening by cyclosporin A (CsA) or Debio-025 (10, 50, 100, and 1,000 nM) measured in mitochondria isolated from sham mouse heart. *P < 0.05 vs. sham.

 

Figure 3
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 3. Inhibition of mPTP opening by postconditioning and Debio-025. A: typical recording of Ca2+-induced mPTP opening in mitochondria isolated from untreated control, postconditioned, and Debio-025-treated hearts. Postconditioned and Debio-025-treated hearts showed more resistance to Ca2+ overload. [Ca2+], Ca2+ concentration. B: inhibition of mPTP opening by postconditioning and Debio-025. CRC was significantly reduced in control vs. sham. Postconditioning and Debio-025 increased CRC. *P < 0.05 vs. control.

 
The in vitro effect of Debio-025 had so far only been assessed in rat brain mitochondria; we then addressed its ability to inhibit transition pore opening assessed using mitochondria isolated from sham hearts. We first performed an in vitro study to determine the capacity of Debio-025 (10–1,000 nM, i.e., from no detectable effect to plateau effect) to inhibit mPTP opening in sham mouse hearts; in this case, mitochondria were isolated from the whole sham LV. CRC was also assessed after 24 h of reperfusion in sham, control, postconditioned, and Debio-025 mice. In all four groups, mitochondria were isolated from the area at risk within the anterior wall of the LV. Care was taken to clearly identify, by visual inspection, the boundaries of the area at risk during ischemia.

Recovery of LV contractile function. At the end of the 30-day period (i.e., just before euthanasia), echocardiography was performed under light anesthesia (ketamine, 80 mg/kg ip). Images were acquired using a 13-MHz linear-array transducer with a digital ultrasound system (Vivid 7, GE Medical Systems). Conventional measurements [LV end-diastolic diameter (LVEDD) and end-systolic diameter (LVESD)] were obtained from gray-scale M-mode traces at the level of the papillary muscles. LV ejection fraction (EF) was calculated from the two-dimensional parasternal long-axis view by the prolate-ellipsoid method (18, 43). After echocardiography, the area at risk was measured using blue dye injection (see above; n = 5 mice/group).

Treatment with Debio-025. Debio-025 (a gift from Debiopharm) is a nonimmunosuppressive cyclosporin that inhibits cyclophilin D within the mitochondrial matrix and has been shown in isolated rat brain mitochondria to be a potent inhibitor of mPTP opening (30). Debio-025 was dissolved in a mixture of Solutol and 94% ethanol (46). Debio-025 was injected over 3 min into the caudal vein at 10 mg/kg 5 min before coronary artery reperfusion.

Statistical Analysis

Comparison between groups was performed using one-way ANOVA. When a significant F value was obtained, means were compared using Tukey's test. Differences in the relationship between infarct size and area at risk were evaluated by analysis of covariance and Tukey's post hoc test, with infarct size as the dependent variable and area at risk as the covariant. Survival was analyzed using Kaplan-Meier curves, and differences among groups were evaluated using the log-rank test. Values are means ± SE. Statistical significance was defined as P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ca2+-Induced Mitochondrial Permeability Transition

In vitro effects of Debio-025. Although Hansson et al. (30) demonstrated that Debio-025 can inhibit mitochondrial permeability transition in isolated rat brain mitochondria, no data were available concerning its effects on cardiac mitochondria. In mitochondria isolated from sham mouse hearts, CRC averaged 336 ± 34 nmol Ca2+/mg mitochondrial proteins (Fig. 2). Debio-025 dose dependently inhibited mPTP opening and significantly increased CRC (482 ± 26 nmol Ca2+/mg mitochondrial proteins) at a concentration as low as 50 nM. It appeared slightly (although not significantly) more potent than the reference mPTP inhibitor CsA (Fig. 2).

CRC. In 21 mice (5 sham, 6 control, 5 postconditioned, and 5 Debio-025), CRC was assessed in mitochondria isolated at the end of the 24-h reperfusion period. In sham hearts, mitochondrial CRC averaged 271 ± 32 nmol Ca2+/mg mitochondrial proteins (Fig. 3). As expected, prolonged ischemia-reperfusion resulted in a significant decrease in CRC: 53 ± 16 nmol Ca2+/mg mitochondrial proteins in control (P < 0.001 vs. sham). Postconditioning and Debio-025 improved CRC: 132 ± 13 and 153 ± 31 nmol Ca2+/mg mitochondrial proteins, respectively [P < 0.05 vs. control, P = not significant (NS) in postconditioning and Debio-025; Fig. 3].

Area at Risk and Infarct Size

Twenty-nine mice (11 control, 9 postconditioned, and 9 Debio-025) were included in the area at risk and infarct size protocol. Three mice died before completion of the experiment: one control, one postconditioned, and one Debio-025. Data are thus presented for the remaining 26 mice: 10 control, 8 postconditioned, and 8 Debio-025.

Area at risk was comparable among the three groups: 41 ± 7, 41 ± 6, and 53 ± 4% of the LV weight in control, postconditioning, and Debio-025 groups, respectively (P = NS among groups). Ischemic postconditioning significantly reduced infarct size: 35 ± 4% vs. 61 ± 6% of the area at risk in the control group (P < 0.05; Fig. 4A). The extent of infarct size reduction induced by Debio-025 was similar to that induced by postconditioning: 32 ± 7% of the risk region (P < 0.05 vs. control, P = NS vs. postconditioning). These results were confirmed when infarct size was plotted vs. its major determinant in the mouse model, i.e., area at risk. As shown in Fig. 4B, most data points for the postconditioning and Debio-025 groups lie below the control line, indicating that, for any size of area at risk, postconditioning and Debio-025 hearts developed significantly smaller infarcts than controls.


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

  		Fig. 4. A: area at risk (AR) and infarct size. All 3 groups displayed comparable AR. Area of necrosis (AN), expressed as a function of area at risk weight, was significantly reduced in postconditioned and Debio-025 groups. *P < 0.05 vs. control. B: infarct size as a function of area at risk. There was a significant correlation between infarct size and area at risk in the control group. Most data points for postconditioned or Debio-025-treated hearts lie below control line, indicating that, for any size of area at risk, these hearts developed significantly smaller infarcts.

 
Recovery of LV Contractile Function

At 30 days post-MI, area at risk was comparable among the three groups: from 29 ± 4% to 32 ± 1% of the LV weight (P = NS among groups). The two-dimensional and M-mode measurements were interpretable in all animals that completed the 30 day follow-up period (5 sham, 10 control, 12 postconditioned, and 13 Debio-025; Fig. 5). Control hearts exhibited a significant LV dilatation and impairment in LV global function (EF; Fig. 6). LVEDD and LVESD averaged 4.2 ± 0.4 and 2.5 ± 0.5 mm, respectively in the control group and 3.6 ± 0.2 and 1.6 ± 0.3 mm, respectively, in the sham group (P < 0.05 for both). Postconditioned hearts displayed a significant reduction in LVEDD and LVESD: 3.9 ± 0.3 and 2.1 ± 0.2 mm, respectively (P < 0.05 vs. control). This reduced LV dilatation was even more pronounced (although not significantly) in Debio-025-treated hearts with LVEDD and LVESD: 3.8 ± 0.3 and 1.8 ± 0.5 mm, respectively (P < 0.05 vs. control).


Figure 5
View larger version (101K):
[in this window]
[in a new window]

  		Fig. 5. Two-dimensional and M-mode echocardiographs of sham, control, postconditioned, and Debio-025-treated mice on day 30. LV dilatation was significantly reduced in postconditioned and Debio-025-treated hearts compared with control.

 

Figure 6
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 6. Recovery of function 30 days after myocardial infarction (MI). At 30 days after MI, echocardiography allowed assessment of LV dimensions and calculation of shortening fraction (SF) and ejection fraction (EF). LVEDD, LV end-diastolic diameter; LVESD, LV end-systolic diameter. *P < 0.05 vs. sham. {dagger}P < 0.05 vs. control.

 
LV function was significantly improved in postconditioned hearts, with a shortening fraction of 46 ± 4% vs. 41 ± 7% in controls and EF of 74 ± 6% vs. 62 ± 12% in controls (P < 0.05). Similar improvement was observed in Debio-025-treated hearts, with a shortening fraction of 54 ± 9% vs. 41 ± 8% in controls (P < 0.05) and EF of 77 ± 6% vs. 62 ± 12% in controls (P < 0.05; Fig. 6). These differences existed, although heart rate was comparable among treated groups and not different from that expected in normal unsedated mice, indicating that anesthesia was light and did not interfere with assessment of contractile function.

30-Day Survival

Fifty-seven mice (20 control, 14 postconditioned, 18 Debio-025, and 5 sham) were followed up for 30 days to address mortality following acute MI. Two mice died during the surgical preparation: one control and one postconditioned. During the 30 day follow-up period, 11 mice (8 controls, 1 postconditioned, and 2 Debio-025) died.

As shown by the Kaplan-Meier curves in Fig. 7, 30 days after acute MI, the survival rate was significantly better in the postconditioned and Debio-025 groups: 92% and 89%, respectively, vs. 58% in the control group (log-rank test, P < 0.05).


Figure 7
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 7. Postinfarction mortality at 30 days shown as Kaplan-Meier curve of 30-day survival rate after acute MI in mice. Postconditioning and Debio-025 significantly improved survival after acute MI.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrates that inhibition of mitochondrial permeability transition at the time of reperfusion by ischemic postconditioning or Debio-025 can enhance recovery of LV contractile function and improve 30-day survival in the mouse model.

Postconditioning dramatically reduced infarct size in the in vivo mouse heart, with average reduction of 44%, which is as pronounced as in other animal species (4, 32, 35, 38, 47, 54). Debio-025, which specifically binds to the mitochondrial cyclophylin D and reduces the sensitivity of the mPTP to Ca2+, reduced infarct size to a similar extent (i.e., 48%). Mitochondria isolated from postconditioned and Debio-025-treated hearts displayed similar enhanced CRC. This finding is consistent with a preliminary report from our group that postconditioning improved CRC of isolated mitochondria and increased the total and free mitochondrial Ca2+ concentration (6). These data further suggest that mitochondrial Ca2+ concentration is likely not the only signal that regulates mPTP opening upon reperfusion. These results are in agreement with previous reports suggesting that mitochondrial permeability transition plays a key role in lethal reperfusion injury and may be a good candidate for future pharmacological postconditioning in patients (27, 31, 36). Because Debio-025 was administered 5 min before reflow, one might question whether it may also have limited ischemia-induced injury. This is, however, unlikely; previous studies using other inhibitors of transition pore opening administered only 1 min before reperfusion showed comparable infarct size limitation (4, 5). It might be questioned whether the increased CRC in the treated groups could simply be the consequence of more viable mitochondria being harvested from less infarcted tissue. This hypothesis appears, however, unlikely for the following reasons. 1) The isolation procedure eliminates dead cardiomyocytes and ruptured mitochondria; the latter fragile organelles are more abundant in the control group, and remaining mitochondria harvested from this group are, in fact, the least damaged. 2) The same final amount of mitochondrial proteins is used in each group. 3) In our previous study using shorter ischemic insult (10 min, i.e., unable to induce any necrosis), pre- and postconditioning increased CRC in a fully viable tissue (i.e., in the absence of potential selection bias during the mitochondria isolation procedure) (3). Although the increased CRC in postconditioned and Debio-025 groups does not demonstrate a causal link between the inhibition of mPTP opening and infarct size reduction, it strongly suggests that it is the case.

After acute MI, a patient's prognosis has been shown to be influenced by recovery of LV function (41, 45). We therefore sought to determine whether inhibition of mPTP opening, by ischemic postconditioning or Debio-025, may alter this predictor of outcome. We have demonstrated for the first time that inhibition of mPTP opening can ameliorate recovery of cardiac contractile function after MI. Persistence of a reduced LV dilatation and enhanced global contractile function at 30 days suggest a likely long-lasting protection. How inhibition of mitochondrial permeability transition might improve functional recovery remains to be determined.

Mitochondrial permeability transition is responsible for the F1F0-ATPase breaking down, rather than building, ATP (13). Inhibition of mPTP opening might therefore preserve energy levels in a population of ischemic but still viable cardiomyocytes, which might eventually result in better resumption of LV contraction. Whether pharmacological inhibition of mPTP opening might have unspecific side effects on LV remodeling cannot be ruled out. However, in contrast to CsA, which binds to the cytosolic cyclophilin A and acts on the calcineurin-nuclear factor of activated T cells (NFAT) pathway, Debio-025 is a cyclophilin D inhibitor that does not act on the calcineurin-NFAT pathway and has no known action on cardiac hypertrophy that would influence cardiac remodeling (28). The role of other mechanisms that play a role in ischemia-reperfusion damage, including altered function of the sarcoplasmic reticulum or spread of injury via gap junctions, cannot be ruled out (21, 42).

Since postconditioning and Debio-025 treatments were performed at one single time point (i.e., reperfusion) and had comparable impact on lethal reperfusion injury, it appears likely that the limitation of LV dilatation and improvement of contractile function correspond to an unspecific consequence of infarct size limitation (51). This is in agreement with clinical reports indicating that peak creatine kinase value, a surrogate marker for infarct size, is an independent predictor of LV remodeling (8).

Another major observation of the present study is that postconditioning and inhibition of the mPTP by Debio-025 significantly reduced the mortality rate within the first 30 days after acute MI. The mechanism of this major outcome remains to be determined. Most deaths in the control group occurred during the first 2 days after reperfusion. This pattern is different from that usually observed in permanent coronary artery ligation studies in mice, in which mortality incidence is more scattered over the 1st wk after infarction (1, 20, 26). In the mouse model of reperfused MI, most early deaths may be due to lethal ventricular arrhythmias or acute heart failure.

The present study does not allow us to determine which of these two types of events was affected by Debio-025 and postconditioning. One cannot exclude the possibility that both treatments had antiarrhythmic effects. Galagudza et al. (19) and Halkos et al. (29) reported that ischemic postconditioning reduces the incidence of ventricular arrhythmias, although the benefit they reported was not statistically significant. Recently, Kloner et al. (39) reported that postconditioning significantly inhibits reperfusion arrhythmias after a 5-min ischemic insult in the rat heart. Although it has recently been proposed that mitochondria may play a role in postischemic arrhythmias, inner membrane depolarization and activation of inner membrane anion channel, rather than opening of the mPTP, appeared involved (2). To our knowledge, there is no evidence in the literature that inhibition of mPTP opening might per se be antiarrhythmic. One may, however, speculate that inhibition of mPTP opening could limit ventricular arrhythmias by limiting cytosolic Ca2+ oscillations and overload or by attenuating ATP breakdown and secondary opening of the sarcolemmal ATP-sensitive K+ channel and proarrhythmic shortening of action potential duration. Early death after reperfusion may also result from acute heart failure. Zhao et al. (54) did not find that postconditioning attenuates myocardial stunning in the dog heart. However, their follow-up of contractile function was limited to the first 3 h after reperfusion, and it cannot be ruled out that postconditioning or Debio-025 may limit acute LV dilatation and prevent acute heart failure, or even the related stretch-induced arrhythmias, within the first 48 h after reperfusion. Further studies are needed to specifically address these important issues.

Limitations of the Study

This study has several limitations. 1) Mortality following acute MI was assessed only over 30 days and in a relatively small cohort of animals. 2) Although Debio-025 and postconditioning provided very comparable benefits in terms of infarct size reduction and improvement in contractile function, as well as in mitochondrial CRC, this does not demonstrate that the mechanism of these effects was the same. Further studies are needed to demonstrate a causal relationship between inhibition of mitochondrial permeability transition and infarct size reduction in postconditioned hearts.

Conclusion

Using a mouse model of reperfused MI, we have demonstrated that ischemic postconditioning and pharmacological inhibition of the mPTP enhanced the recovery of LV contractile function and improved survival. Since recent reports indicate that postconditioning can protect the human heart, the present data represent an encouraging background for the search for new pharmacological agents aimed at inhibiting the mPTP at the time of reperfusion in patients undergoing acute MI.


   ACKNOWLEDGMENTS
 
L. Gomez was a recipient of a grant from the "Region Rhône-Alpes."


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Ovize, Inserm E0226-Laboratoire de Physiologie Lyon-Nord, 8, Ave. Rockefeller, 69373 Lyon, France (e-mail: ovize{at}sante.univ-lyon1.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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Adachi Y, Saito Y, Kishimoto I, Harada M, Kuwahara K, Takahashi N, Kawakami R, Nakanishi M, Nakagawa Y, Tanimoto K, Saitoh Y, Yasuno S, Usami S, Iwai M, Horiuchi M, Nakao K. Angiotensin II type 2 receptor deficiency exacerbates heart failure and reduces survival after acute myocardial infarction in mice. Circulation 107: 2406–2408, 2003.[Abstract/Free Full Text]
  2. Akar FG, Aon MA, Tomaselli GF, O'Rourke B. The mitochondrial origin of postischemic arrhythmias. J Clin Invest 115: 3527–3535, 2005.[CrossRef][ISI][Medline]
  3. Argaud L, Gateau-Roesch O, Chalabreysse L, Gomez L, Loufouat J, Thivolet-Bejui F, Robert D, Ovize M. Preconditioning delays Ca2+-induced mitochondrial permeability transition. Cardiovasc Res 61: 115–122, 2004.[Abstract/Free Full Text]
  4. 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]
  5. 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]
  6. Argaud L, Gateau-Roesch O, Couture-Lepetit E, Ovize M. Role of matrix calcium accumulation in mitochondrial permeability transition in postconditioned versus preconditioned myocardium (Abstract). Circulation 112, Suppl 3: 310, 2005.
  7. Bernardi P. Mitochondrial transport of cations: channels, exchangers, and permeability transition. Physiol Rev 79: 1127–1155, 1999.[Abstract/Free Full Text]
  8. Bolognese L, Neskovic AN, Parodi G, Cerisano G, Buonamici P, Santoro GM, Antoniucci D. Left ventricular remodeling after primary coronary angioplasty: patterns of left ventricular dilation and long-term prognostic implications. Circulation 106: 2351–2357, 2002.[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. Burns RJ, Gibbons RJ, Yi Q, Roberts RS, Miller TD, Schaer GL, Anderson JL, Yusuf S. The relationships of left ventricular ejection fraction, end-systolic volume index and infarct size to six-month mortality after hospital discharge following myocardial infarction treated by thrombolysis. J Am Coll Cardiol 39: 30–36, 2002.[Abstract/Free Full Text]
  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][ISI][Medline]
  12. Crompton M. Mitochondrial intermembrane junctional complexes and their role in cell death. J Physiol 529: 11–21, 2000.[Abstract/Free Full Text]
  13. 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.[ISI][Medline]
  14. Darling CE, 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]
  15. 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]
  16. Duchen MR, McGuinness O, Brown LA, Crompton M. On the involvement of a cyclosporin A-sensitive mitochondrial pore in myocardial reperfusion injury. Cardiovasc Res 27: 1790–1794, 1993.[Free Full Text]
  17. 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 3beta. Anesthesiology 103: 987–995, 2005.[CrossRef][ISI][Medline]
  18. Folland ED, Parisi AF, Moynihan PF, Jones DR, Feldman CL, Tow DE. Assessment of left ventricular ejection fraction and volumes by real-time, two-dimensional echocardiography. A comparison of cineangiographic and radionuclide techniques. Circulation 60: 760–766, 1979.[Abstract/Free Full Text]
  19. Galagudza M, Kurapeev D, Minasian S, Valen G, Vaage J. Ischemic postconditioning: brief ischemia during reperfusion converts persistent ventricular fibrillation into regular rhythm. Eur J Cardiothorac Surg 25: 1006–1010, 2004.[Abstract/Free Full Text]
  20. Gao XM, Dart AM, Dewar E, Jennings G, Du XJ. Serial echocardiographic assessment of left ventricular dimensions and function after myocardial infarction in mice. Cardiovasc Res 45: 330–338, 2000.[Abstract/Free Full Text]
  21. Garcia-Dorado D, Inserte J, Ruiz-Meana M, Gonzalez MA, Solares J, Julia M, Barrabes JA, Soler-Soler J. Gap junction uncoupler heptanol prevents cell-to-cell progression of hypercontracture and limits necrosis during myocardial reperfusion. Circulation 96: 3579–3586, 1997.[Abstract/Free Full Text]
  22. Gheorghiade M, Bonow RO. Chronic heart failure in the United States: a manifestation of coronary artery disease. Circulation 97: 282–289, 1998.[Free Full Text]
  23. Gomez L, Chavanis N, Argaud L, Chalabreysse L, Gateau-Roesch O, Ninet J, Ovize M. Fas-independent mitochondrial damage triggers cardiomyocyte death following ischemia-reperfusion. Am J Physiol Heart Circ Physiol 289: H2153–H2158, 2005.[Abstract/Free Full Text]
  24. Gomez L, Raisky O, Chalabreysse L, Verschelde C, Bonnefoy-Berard N, Ovize M. Link between immune cell infiltration and mitochondria-induced cardiomyocyte death during acute cardiac graft rejection. Am J Transplant 6: 487–495, 2006.[CrossRef][ISI][Medline]
  25. Gornall AGB, Charles J, David Maxima M. Determination of serum proteins by means of the biuret reaction. J Biol Chem 177: 751–766, 1949.[Free Full Text]
  26. Gould KE, Taffet GE, Michael LH, Christie RM, Konkol DL, Pocius JS, Zachariah JP, Chaupin DF, Daniel SL, Sandusky GE Jr, Hartley CJ, Entman ML. Heart failure and greater infarct expansion in middle-aged mice: a relevant model for postinfarction failure. Am J Physiol Heart Circ Physiol 282: H615–H621, 2002.[Abstract/Free Full Text]
  27. Griffiths EJ, Halestrap AP. Mitochondrial non-specific pores remain closed during cardiac ischaemia, but open upon reperfusion. Biochem J 307: 93–98, 1995.[ISI][Medline]
  28. Griffiths EJ, Halestrap AP. Protection by cyclosporin A of ischemia/reperfusion-induced damage in isolated rat hearts. J Mol Cell Cardiol 25: 1461–1469, 1993.[CrossRef][ISI][Medline]
  29. Halkos ME, Kerendi F, Corvera JS, Wang NP, Kin H, Payne CS, Sun HY, Guyton RA, Vinten-Johansen J, Zhao ZQ. Myocardial protection with postconditioning is not enhanced by ischemic preconditioning. Ann Thorac Surg 78: 961–969, 2004.[Abstract/Free Full Text]
  30. Hansson MJ, Mattiasson G, Mansson R, Karlsson J, Keep MF, Waldmeier P, Ruegg UT, Dumont JM, Besseghir K, Elmer E. The nonimmunosuppressive cyclosporin analogs NIM811 and UNIL025 display nanomolar potencies on permeability transition in brain-derived mitochondria. J Bioenerg Biomembr 36: 407–413, 2004.[CrossRef][ISI][Medline]
  31. 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]
  32. Heusch G, Buchert A, Feldhaus S, Schulz R. No loss of cardioprotection by postconditioning in connexin 43-deficient mice. Basic Res Cardiol 101: 354–356, 2006.[CrossRef][Medline]
  33. Ho KK, Anderson KM, Kannel WB, Grossman W, Levy D. Survival after the onset of congestive heart failure in Framingham Heart Study subjects. Circulation 88: 107–115, 1993.[Abstract/Free Full Text]
  34. Ichas F, Jouaville LS, Sidash 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][ISI][Medline]
  35. Iliodromitis EK, Georgiadis M, Cohen MV, Downey JM, Bofilis E, Kremastinos DT. Protection from postconditioning depends on the number of short ischemic insults in anesthetized pigs. Basic Res Cardiol 101: 502–507, 2006.[CrossRef][ISI][Medline]
  36. Javadov SA. Ischaemic preconditioning inhibits opening of mitochondrial permeability transition pores in the reperfused rat heart. J Physiol 549: 513–524, 2003.[Abstract/Free Full Text]
  37. Kin H, Zatta AJ, Lofye MT, Amerson BS, Halkos ME, Kerendi F, Zhao ZQ, Guyton RA, Headrick JP, Vinten-Johansen J. Postconditioning reduces infarct size via adenosine receptor activation by endogenous adenosine. Cardiovasc Res 67: 124–133, 2005.[Abstract/Free Full Text]
  38. 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]
  39. Kloner RA, Dow J, Bhandari A. Postconditioning markedly attenuates ventricular arrhythmias after ischemia-reperfusion. J Cardiovasc Pharmacol Ther 11: 55–63, 2006.[Abstract/Free Full Text]
  40. Massie BM, Shah NB. Evolving trends in the epidemiologic factors of heart failure: rationale for preventive strategies and comprehensive disease management. Am Heart J 133: 703–712, 1997.[CrossRef][ISI][Medline]
  41. Pfeffer MA, Braunwald E. Ventricular remodeling after myocardial infarction. Experimental observations and clinical implications. Circulation 81: 1161–1172, 1990.[Abstract/Free Full Text]
  42. Piper HM, Kasseckert S, Abdallah Y. The sarcoplasmic reticulum as the primary target of reperfusion protection. Cardiovasc Res 70: 170–173, 2006.[Free Full Text]
  43. Rodrigues AC, Hataishi R, Ichinose F, Bloch KD, Derumeaux G, Picard MH, Scherrer-Crosbie M. Relationship of systolic dysfunction to area at risk and infarction size after ischemia-reperfusion in mice. J Am Soc Echocardiogr 17: 948–953, 2004.[CrossRef][ISI][Medline]
  44. Sharpe N, Doughty R. Epidemiology of heart failure and ventricular dysfunction. Lancet 352 Suppl 1: SI3–SI7, 1998.[CrossRef][Medline]
  45. St. John Sutton M, Pfeffer MA, Plappert T, Rouleau JL, Moye LA, Dagenais GR, Lamas GA, Klein M, Sussex B, Goldman S, et al. Quantitative two-dimensional echocardiographic measurements are major predictors of adverse cardiovascular events after acute myocardial infarction: the protective effects of captopril. Circulation 89: 68–75, 1994.[Abstract/Free Full Text]
  46. Strickley RG. Solubilizing excipients in oral and injectable formulations. Pharm Res 21: 201–230, 2004.[CrossRef][ISI][Medline]
  47. Tang XL, Sato H, Tiwari S, Dawn B, Bi Q, Li Q, Shirk G, Bolli R. Cardioprotection by postconditioning in conscious rats is limited to coronary occlusions <45 min. Am J Physiol Heart Circ Physiol 291: H2308–H2317, 2006.[Abstract/Free Full Text]
  48. 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]
  49. 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]
  50. 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]
  51. Yang F, Liu YH, Yang XP, Xu J, Kapke A, Carretero OA. Myocardial infarction and cardiac remodelling in mice. Exp Physiol 87: 547–555, 2002.[Abstract]
  52. 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]
  53. 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]
  54. 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]
  55. Zoratti M, Szabo I. The mitochondrial permeability transition. Biochim Biophys Acta 1241: 139–176, 1995.[Medline]



This article has been cited by other articles:


Home page
 NEJMHome page
D. J. Hausenloy and D. M. Yellon
Time to Take Myocardial Reperfusion Injury Seriously
N. Engl. J. Med., July 31, 2008; 359(5): 518 - 520.
[Full Text] [PDF]

Home page
 CirculationHome page
L. Gomez, M. Paillard, H. Thibault, G. Derumeaux, and M. Ovize
Inhibition of GSK3{beta} by Postconditioning Is Required to Prevent Opening of the Mitochondrial Permeability Transition Pore During Reperfusion
Circulation, May 27, 2008; 117(21): 2761 - 2768.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/3/H1654    most recent
01378.2006v1
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 (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gomez, L.
Right arrow Articles by Ovize, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gomez, L.
Right arrow Articles by Ovize, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.