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Am J Physiol Heart Circ Physiol 291: H2265-H2271, 2006. First published June 23, 2006; doi:10.1152/ajpheart.00209.2006
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Controlled reperfusion after hypothermic heart preservation inhibits mitochondrial permeability transition-pore opening and enhances functional recovery

J. C. Bopassa,1 David Vandroux,2 M. Ovize,1 and R. Ferrera1

1Faculté de Médecine Lyon-Nord, Université Claude Bernard Lyon-1, Institut National de la Santé et de la Recherche Médicale E 0226, Lyon; and 2Université de Bourgogne, Dijon, France

Submitted 27 February 2006 ; accepted in final form 16 June 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We investigated whether low-pressure reperfusion may attenuate postischemic contractile dysfunction, limits necrosis and apoptosis after a prolonged hypothermic ischemia, and inhibits mitochondrial permeability transition-pore (MPTP) opening. Isolated rats hearts (n = 72) were exposed to 8 h of cold ischemia and assigned to the following groups: 1) reperfusion with low pressure (LP = 70 cmH2O) and 2) reperfusion with normal pressure (NP = 100 cmH2O). Cardiac function was assessed during reperfusion using the Langendorff model. Mitochondria were isolated, and the Ca2+ resistance capacity (CRC) of the MPTP was determined. Malondialdehyde (MDA) production, caspase-3 activity, and cytochrome c were also assessed. We found that functional recovery was significantly improved in LP hearts with rate-pressure product averaging 30,380 ± 1,757 vs. 18,000 ± 1,599 mmHg/min in NP hearts (P < 0.01). Necrosis, measured by triphenyltetrazolium chloride staining and creatine kinase leakage, was significantly reduced in LP hearts (P < 0.01). The CRC was increased in LP heart mitochondria (P < 0.01). Caspase-3 activity, cytochrome c release, and MDA production were reduced in LP hearts (P < 0.001 and P < 0.01). This study demonstrated that low-pressure reperfusion after hypothermic heart ischemia improves postischemic contractile dysfunction and attenuates necrosis and apoptosis. This protection could be related to an inhibition of mitochondrial permeability transition.

mitochondria; apoptosis

RECENT EVIDENCE INDICATES that lethal reperfusion injury occurs at the time of reperfusion after normothermic is che mia. Mitochondrial permeability transition appears to be a pivotal event in cell death, after is che mia-reperfusion (43). Mitochondrial permeability transition results in the opening of a large pore in the inner mitochondrial membrane, whose structure remains incompletely known. Mitochondrial permeability transition-pore (MPTP) opening is related with uncoupling of the respiratory chain, efflux of Ca2+ and small proteins such as the proapop totic cytochrome c, and matrix swelling (4, 28). MPTP opening is mostly induced by matrix Ca2+ accumulation, adenine nucleotide depletion, increased inorganic phosphate concentration, and oxidative stress, all features of is che mia-reperfusion. Griffiths and Halestrap (16) showed that the MPTP is closed during is che mia but opens in the early minutes of reperfusion. Hausenloy et al. (20) proposed that pharmacological and is che mic preconditioning could act at the time of reperfusion by inhibiting MPTP opening. These authors also proposed that the inhibition of MPTP opening during the reperfusion could exert a myocardial protection (19). Our group also confirmed the crucial role played by MPTP during pre- (1) and postconditioning (2).

Many studies performed using animal preparations or clinical models attempted to modify the conditions of reperfusion to attenuate its deleterious effects. Controlled reperfusion after an is che mic insult has been proposed to protect the reperfused myocardium (21, 26, 32, 38).

Our group recently demonstrated that a low-pressure reperfusion might protect the heart at the time of reperfusion after an irreversible normothermic is che mic insult (6). Furthermore, the protection afforded with a low-pressure reperfusion seems to act by the same route (phosphatidylinositol 3-kinase-Akt pathway) as postconditioning (5).

However, the protective effect of controlled reperfusion after a hypothermic cardioplegic arrest remains debated (13, 34–36). Whether a similar mechanism applies during reperfusion after a hypothermic is che mia is currently unknown. During cold preservation, damage to isolated hearts depends on numerous factors, including the temperature, the pH, the composition of the cardioplegia, and/or the preservative solutions.

Therefore, the objective of this study was to determine whether a controlled low-pressure reperfusion could protect against cellular necrosis and apop to sis by acting on MPTP opening and improve postis che mic contractile dysfunction after a prolonged hypothermic is che mia.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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Surgical Procedure

The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (National Institutes of Health Publication No. 85-23, Revised 1996).

Male Wistar rats, weighing 350–450 g, were anesthetized with pentobarbital sodium (50 mg/kg). Heparin (200 IU/kg) was injected in the femoral vein. Hearts were quickly removed, the aorta was cannulated, and cold Celsior solution was perfused during 1 min to arrest hearts.

Experimental Design

Hearts were then immersed in the same hypothermic solution during 8 h at 4°C (is che mic period). Two different protocols were performed.

Protocol I was applied to evaluate functional recovery and tissue necrosis after 8 h of global cold is che mia at 4°C and 60 min of reperfusion. Reperfusion was performed by using a Langendorff system with Krebs-Henseleit bicarbonate buffer [containing (in mmol/l) 11.0 glucose, 118.5 NaCl, 4.75 KCl, 1.19 MgSO4, 1.18 KH2PO4, 25.0 NaHCO3, and 1.4 CaCl2] at pH 7.4. The buffer was bubbled with 95% O2-5% CO2 at 37°C. The left ventricle (LV) was paced at a constant rate of 300 beats/min.

Protocol II was used to assess the Ca2+ resistance capacity (CRC) of the MPTP and to measure cytochrome c release, caspase-3 activity (indicators of apop to sis), and myocardial malondialdehyde (MDA) production (an index of lipid peroxidation by oxygen-derived free radicals).

Protocol I. One group of hearts underwent no intervention for the whole duration of the experiment (control, n = 6). All other hearts underwent 8 h of global hypothermic (4°C) ischemia followed by reperfusion. Animals were randomly assigned to one of the two following groups (n = 6–8/group): NP group (normal pressure): myocardium was reperfused at normal pressure (i.e., 100 cmH2O) after the cold ischemic storage and LP group (low pressure): myocardium was reperfused at a pressure of 70 cmH2O.

These two perfusion pressures were obtained by adjusting the higher of the perfusion column. It can be noted that 100 cmH2O is considered as a normal perfusion pressure for a rat heart under physiological conditions.

After 60 min reperfusion using the two perfusion pressures, hearts were reperfused an additional 15 min. These last 15 min corresponded to the return to normal pressure in LP hearts. Functional recovery was analyzed throughout the reperfusion, whereas myocardial necrosis [TTC staining, creatine kinase (CK) and lactate dehydrogenase (LDH) release] was assessed at the end of reperfusion (75 min). We chose this short period of reperfusion because we found on previous experiments that extending the duration of reperfusion to 120 min in our model did not change either the absolute values of infarct size or the difference between the two groups. It also did not change enzymatic release (CK, LDH) and functional recovery (unpublished data).

Protocol II. All hearts underwent 8 h of global hypothermic (4°C) ischemia followed by 10 min of reperfusion. Animals were randomly assigned to one of the two previously defined groups (n = 6/group). At the end of the 10-min reperfusion, hearts were excised for measurement of CRC, MDA content, cytochrome c release, and caspase-3 activity.

Analysis

Functional recovery. The LV systolic pressure (LVSP) and the LV end-diastolic pressure (LVEDP) were measured using a latex balloon introduced in the LV and expanded to exert a physiological end-diastolic pressure of 5 mmHg. The rate-pressure product [RPP = (LVSP – LVEDP) x heart rate], the rate of systolic contraction (dP/dtmax), and diastolic relaxation (–dP/dtmin) were calculated. Coronary flow was measured by timed collections of the coronary effluent flow.

Myocardial necrosis. Cellular injury was evaluated by both measurements of CK and LDH release in the coronary effluent at the end of the reperfusion period (Coulter kit; Beckman, Galway, Ireland). Enzyme leakage measurements were corrected by taking into account the coronary flow rate and the weight of the heart. Myocardial necrosis was also assessed using triphenyltetrazolium chloride (TTC) staining, as previously described (41). Briefly, the heart was cut into five transverse slices, parallel to the atrioventricular groove. After the right ventricular tissue was removed, heart slices were weighed and incubated for 20 min in a 1% solution of TTC at 37°C to differentiate infarcted (pale) from viable (brick red) myocardial area. The slices were then photographed. The extent of the area of necrosis was quantified by computerized planimetry. Total area of necrosis was then calculated and expressed as a percentage of total LV area.

Ca2+-Induced Mitochondrial Permeability Transition

Preparation of isolated mitochondria. Preparation of mitochondria was adapted from a previously described procedure (14). All operations were carried out in the cold. Myocardial sections (~1 g) were placed in isolation buffer A containing (in mM) 70 sucrose, 210 mannitol, and 1 EDTA in 50 Tris·HCl, pH 7.4. The tissue was finely minced with scissors and then homogenized in the same buffer (1 ml buffer/g tissue) using, successively, a Kontes tissue grinder and a Potter Elvejem homogenizer. The homogenate was centrifuged at 1,300 g for 3 min. The supernatant was poured through cheesecloth and centrifuged at 10,000 g for 10 min. The supernatant was collected and stored at –80°C for subsequent cytochrome c and caspase-3 activity measurements. The mitochondrial pellet was resuspended in isolation buffer B containing (in mM) 70 sucrose, 210 mannitol, and 0.1 EDTA in 50 Tris·HCl, pH 7.4. After aliquots were removed for protein measurements, the mitochondria (by aliquots of 5-mg proteins) were washed in isolation buffer B, centrifuged at 6,800 g for 10 min, and stored as pellets on ice before Ca2+-induced MPTP opening experiments. Protein content was routinely assayed according to Gornall's procedure using BSA as a standard (15).

Ca2+-induced MPTP opening. CRC of the MPTP was assessed after in vitro Ca2+ overload. Isolated mitochondria (5 mg proteins) were suspended in 100 µl buffer B and added to 900 µl of buffer C (in mM: 150 sucrose, 50 KCl, 2 KH2PO4, and 5 succinic acid in 20 Tris·HCl, pH 7.4) within a Teflon chamber equipped with a Ca2+-specific microelectrode, in conjunction with a reference electrode. Modifications of the medium (i.e., extramitochondrial) Ca2+ concentration were continuously recorded using custom-made Synchronie software. Mitochondria were stirred gently for 1.5 min. At the end of the preincubation period, 20 µM CaCl2 were added every 60 s. Each 20 µM CaCl2 administration is recorded as a peak of extramitochondrial Ca2+ concentration. Ca2+ is then rapidly taken up by the mitochondria, resulting in a return of extramitochondrial Ca2+ concentration to near baseline level. After sufficient Ca2+ loading, extramitochondrial Ca2+ concentration abruptly increases, indicating a massive release of Ca2+ by mitochondria because of MPTP opening, as previously shown (1, 2). The amount of Ca2+ necessary to trigger this massive Ca2+ release is used here as an indicator of the CRC of the mitochondrial permeability transition.

MDA production. After 10 min of reperfusion, biopsies were harvested from the LV and quickly frozen. The MDA level was determined by HPLC using the thiobarbituric acid test and expressed as picomoles per milligram of protein, as previously described (6).

Caspase-3 activity. The supernatant obtained from mitochondria centrifugation was collected. Aliquots containing 80 µg of supernatant proteins were incubated in 50 µl of buffer A [25 mmol/l HEPES, 5 mmol/l MgCl2, 1 mmol/l EGTA, 10 µg/ml leupeptin, and 1 mmol/l phenylmethylsulfonyl fluoride (PMSF), pH 7.5]. The previous mixture was diluted with 225 µl of freshly prepared buffer B [25 mmol/l HEPES, 0.1% (wt/vol) 3-(cholamidopropyl dimethylammoniol)-1-propane sulfonate, 10 mmol/l dithiothreitol, 100 U/ml aprotinin, and 1 mmol/l PMSF, pH 7.5] containing 167 µmol/l substrate caspase-3 inhibitor DEVD and incubated for 60 min at 37°C. Fluorescence was measured using an excitation wavelengh of 342 nm and emission wavelength of 441 nm. Standards containing 0–3,000 pmol of aminomethylcoumarin (AMC) were used to determine the amount of fluorochrome released.

Cytochrome c. Total protein was quantified according to the Bradford method. Thirty micrograms of protein were loaded per lane on 16% SDS-PAGE. Migration and electroblotting on nylon membranes were done according to the manufacturer's instructions (Bio-Rad, Hercules, CA). Membranes were blocked for 60 min at room temperature with 5% nonfat milk in Tris-buffered saline (in mM: 25 Tris, 137 NaCl, and 2.7 KCl) containing 0.1% Tween 20 and incubated with anti-cytochrome c antibody (Pharmingen, San Diego, CA) at 1:1,000 dilution for 120 min in the same buffer with gentle agitation. After three washes, the blots were incubated for 60 min with 1:10,000 dilution of peroxidase-conjugated donkey anti-mouse IgG. After being further washed, the immunocomplexes were visualized using an enhanced chemiluminescence system (Amersham, Little Chalfort, UK).

Statistics

Statistical comparisons were performed using the ANOVA and Fischer protected least significant difference test. All results are expressed as means ± SE. A P value <0.05 was considered as indicative of a statistically significant difference.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Protocol I: Myocardial Necrosis and Functional Recovery After Ischemia and Reperfusion

Functional recovery. In the NP group, baseline RPP averaged 38,000 ± 2,000 mmHg/min. During the reperfusion period, recovery of RPP was impaired, ranging from 11,375 ± 1,016 mmHg/min at 10 min to 18,000 ± 1,599 mmHg/min at 60 min (P < 0.001 vs. baseline and control; Fig. 1). After 60 min of reperfusion, mean LV dP/dtmax and LV dP/dtmin were significantly decreased, averaging 1,270 ± 69 and 8,43 ± 90 mmHg/s, respectively (P < 0.001 vs. control). Mean CF was unchanged in NP hearts compared with control values (Table 1).


Figure 1
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  		Fig. 1. Postischemic functional recovery. Effects of low pressure (LP, 70 cmH2O) and normal pressure (NP, 100 cmH2O) during reperfusion of cold ischemic hearts on the rate-pressure product (RPP). Values shown are means of 6 values ± SE. {dagger}P < 0.05 and {dagger}{dagger}P < 0.01, different from LP group value.

 

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  		Table 1. Hemodynamics and functional recovery at 60 min of reperfusion

 
In the LP group, baseline RPP was comparable to that of the NP group. During the reperfusion period, RPP was significantly higher than in the NP group at all times, being, e.g.: 22,316 ± 1,825 mmHg/min at 10 min and 24,316 ± 1,758 mmHg/min at 60 min (P < 0.05 vs. NP; Fig. 1). Both mean LV dP/dtmax and LV dP/dtmin of hearts in the LP groups were significantly higher than in NP hearts (P < 0.01) despite a reduced mean coronary flow (Table 1). Return to normal perfusion pressure at the end of the experiment slightly increased RPP in the LP group to reach 30,380 ± 1,757 mmHg/min (P < 0.01 vs. NP). During this last period, the coronary flow of LP hearts increased to values similar to those of NP hearts (averaging 13.8 ± 1 and 14.0 ± 1.4 ml·min–1·g–1, respectively, for LP and NP hearts).

Myocardial necrosis. CK release was significantly reduced in the LP vs. NP group, averaging 21 ± 12 vs. 90 ± 16 IU/l at 60 min of reperfusion (P < 0.01; Fig. 2). LDH release was similarly reduced in the LP vs. NP group, averaging 80 ± 21 and 137 ± 44 UI/l at 60 min of reperfusion, respectively (P < 0.05; Fig. 2). TTC staining revealed that infarct size averaged 6.5 ± 2% of LV weight in LP vs. 15 ± 3% in NP (P < 0.01; Fig. 3).


Figure 2
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  		Fig. 2. Creatine kinase (CPK) and lactate dehydrogenase (LDH) release. Enzymatic release during reperfusion in the coronary effluent during of hearts reperfused at NP (100 cmH2O) and LP (70 cmH2O). Values shown are means of 6 values ± SE. ***P < 0.001, different from control value. {dagger}P < 0.05 and {dagger}{dagger}P < 0.01, different from LP group value.

 

Figure 3
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  		Fig. 3. Infarct size evaluated by triphenyltetrazolium chloride staining. LP significantly reduced infarct size vs. NP group. Values shown are means of 6 values ± SE. ***P < 0.001, different from control value. LV, left ventricular. {dagger}{dagger}P < 0.01, different from LP group value.

 
Protocol II: Ca2+-Induced Mitochondrial Permeability Transition and Apoptosis

CRC. The effect of 10 min of controlled reperfusion after 8 h of cold ischemia on MPTP opening is depicted in Fig. 4. In the control group, the amount of Ca2+ required to open the MPTP averaged 372 ± 14 µmol. The CRC was significantly reduced in the NP group to 156 ± 38 µmol/5 µmol proteins (P < 0.001 vs. control). In the LP group, the Ca2+ load required to open MPTP was significantly higher than in NP hearts, averaging 272 ± 19 µmol/5 µmol proteins (P < 0.01 vs. NP group).


Figure 4
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  		Fig. 4. Ca2+-induced mitochondrial permeability transition-pore (MPTP) opening. Final quantity of extramitochondrial Ca2+ addition [µmol/5 mg protein (prot)] from hearts reperfused 10 min at LP (70 cmH2O) and NP (100 cmH2O) pressure after 8 h of global cold ischemia. Values shown are means of 6 values ± SE. *P < 0.05 and ***P < 0.001, different from control value. {dagger}{dagger}P < 0.01, different from LP group value. Inset: example of typical recording of MPTP opening in isolated mitochondria obtained from one NP and one LP heart. In the NP mitochondria, a Ca2+ overload of 156 µM (mean of 8 pulses of 20 µM) was required to induce MPTP opening. In LP mitochondria, a significantly higher Ca2+ overload (272 µM, mean of 14 pulses) was required to induce MPTP opening. Vertical arrows indicate the first administration of 20 µM of Ca2+ in the NP or LP mitochondria suspension. Each spike of Ca2+ concentration results from a repeated administration of 20 µM of Ca2+.

 
MDA production. Myocardial MDA content assessed after 8 h of cold ischemia and 10 min of reperfusion are reported in Fig. 5. Myocardial MDA content was significantly increased in the NP group (69 ± 12 pmol/mg protein) compared with shams. LP hearts displayed a reduced MDA content that averaged 45 ± 3 pmol/mg protein (P < 0.05 vs. NP, P = not significant vs. control).


Figure 5
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  		Fig. 5. Myocardial malondialdehyde (MDA). MDA levels of hearts reperfused 10 min at LP (70 cmH2O) and NP (100 cmH2O). Values shown are means of 6 values ± SE. **P < 0.01, different from control and LP values.

 
Cytochrome c release. Extramitochondrial cytochrome c was absent in control hearts (Fig. 6). LP significantly reduced cytochrome release compared with NP.


Figure 6
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  		Fig. 6. Cytochrome c (Cyt C) release. Cytosolic cytochrome c release of hearts reperfused 10 min at LP (70 cmH2O) and NP (100 cmH2O). Values shown are means of 6 values ± SE. ***P < 0.001, different from control values. {dagger}{dagger}{dagger}P < 0.001, different from LP group value. Top: example of Western blotting analysis of cytochrome c for 2 hearts in each group.

 
Caspase-3 activity. Caspase-3 activity was dramatically increased in NP, averaging 2,360 ± 259 pmol AMC·h–1·mg–1 at 10 min of reperfusion (P < 0.001 vs. control; Fig. 7). LP hearts displayed a reduced caspase-3 activity that averaged 606 ± 71 AMC·h–1·mg–1 (P < 0.001 vs. NP, P < 0.05 vs. control).


Figure 7
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  		Fig. 7. Caspase-3 activity. Activity of caspase-3 of hearts reperfused 10 min at LP (70 cmH2O) and NP (100 cmH2O). AMC, aminomethylcoumarin. Values shown are means of 6 values ± SE. *P < 0.05 and ***P < 0.001, different from control values. {dagger}{dagger}{dagger}P < 0.001, different from LP group value.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Lethal reperfusion injury after a prolonged ischemic insult has been a matter of debate over the past two decades. Recent evidence clearly indicated that irreversible myocardial damage occurs during reperfusion, as demonstrated by the existence of a postconditioning-induced cardioprotection (42). This implies that the conditions of reperfusion are determinant for myocardial viability after a sustained ischemia. Previous studies conducted by Buckberg (9) and by Vinten-Johansen et al. (39) investigated the role of several factors modifying the conditions of reperfusion and the composition of the reperfusate. Kaneda et al. (23) demonstrated, in the rat model, that reducing PO2 (300 mmHg) during early reperfusion improves contractile function after 30 min of normothermic ischemia. In contrast, high PO2 (700 mmHg) leads to myocardial damage. Di Venosa et al. (11) observed that low PO2 reperfusion (150 mmHg) after 45 min of ischemia partially reduced diastolic function but failed to improve cardiac contractility. In a previous study, our group demonstrated that low-pressure reperfusion protects isolated rat hearts submitted to 40 min of warm ischemia (37°C) and 60 min of reperfusion (6). Furthermore, we recently showed that both low-pressure reperfusion and postconditioning activated the same key phosphatidylinositol 3-kinase-Akt pathway, inducing inhibition of MPTP opening at the initiation of reperfusion (5).

However, the possibility that controlled reperfusion may improve functional recovery after a prolonged global hypothermic ischemia remains unclear, and discrepancies appear in the literature (10, 35). Low-pressure reperfusion was successfully used on rat and pig lung after cold preservation (18, 29, 37) and allowed our group to transplant human cardiac grafts after a prolonged cold ischemia ranging from 10 to 13 h (31). On the other hand, Rabinov et al. (34) reported that low-pressure reperfusion after cold storage produced subendocardial ischemia and exacerbated ventricular fibrillation in canine hearts. Rosenfeldt et al. (36) failed to improve functional recovery by using various reperfusion pressures (between 40 and 100 mmHg) on patients submitted to coronary bypass grafting. Fontan et al. (13) described an ambivalent effect of low-pressure reperfusion (50 mmHg) on patients undergoing coronary bypass.

Why would there be a difference of efficiency of the low-pressure procedure after warm and cold ischemia? One may hypothesize that the procedure of cardioplegic arrest may be determinant to explain the discrepancy. In a preliminary study of our group, we failed to protect isolated rat heart submitted to 8 and 12 h cold ischemia in high-K+ cardioplegic solution and reperfused with low-pressure reperfusion (unpublished data). We might suppose that, submitted to high-potassium cardioplegia, endothelial cells would be impaired and produce inappropriate nitric oxide (40). Nitric oxide at physiological levels is known to protect the cell by preserving ATP, preventing MPTP opening and limiting apoptosis (22, 24, 30). Conversely, inadequate nitric oxide production would induce 1) negative inotrope effect, as previously shown (27, 33) and 2) activation of MPTP opening (nitric oxide) is a potent activator of MPTP (8).

In the present study, we showed that, after cold cardioplegia induced with Celsior solution and 8 h of global hypothermic ischemia, significant reperfusion injury occurred with uncontrolled reperfusion. Applying a low pressure in the early period of reperfusion blunted this irreversible damage. A notable point is that this protection was efficient as soon as 10 min after reflow and persisted when normal pressure was set back 1 h later, strongly suggesting that low pressure prevents irreversible myocardial injury by acting during the early minutes of reperfusion (Fig. 1).

Low-pressure reperfusion also results in a reduced coronary flow (Table 1). Kin et al. (25) recently proposed an interesting hypothesis. They suggested that the protection induced by postconditioning could be because of a delayed washout of intravascular adenosine (and increased retention time of adenosine in the extracellular space), associated with a reduced coronary flow during the first 2 min of reperfusion. In the present study, the 35% reduction of coronary flow induced by controlled reperfusion is in accordance with this hypothesis. Moreover, initial low-flow reperfusion may reduce oxygen influx and consequently limit free radical production. Indeed, ischemic hearts reperfused at low pressure exhibited a significantly reduced MDA content, suggesting a reduced lipid peroxidation via reactive oxygen species (Fig. 5). The reduction of coronary flow may also prevent Ca2+ overload after normothermic ischemia in the pig heart, as we previously showed (12). Free radical and Ca2+ overload are two major regulators of MPTP. We thus investigated whether MPTP opening might be associated with controlled reperfusion after cold ischemia. MPTP opening is indeed a crucial event in cardiomyocyte death after ischemia-reperfusion (16, 20, 28). MPTP opening at the onset of reperfusion after a prolonged ischemia is controlled by many factors, especially matrix Ca2+ accumulation and/or a production of free radicals. MPTP opening results in cell death. Recent papers point out the crucial role of MPTP in necrosis of the heart and brain rather than apoptosis (17). Moreover, cyclophilin D knockout mice are protected against necrosis but not against apoptosis after an ischemia-reperfusion sequence (3). Other reports showed the role of MPTP in apoptosis induced by myocardial ischemia. It was shown that opening the transition pore of the inner mitochondrial membrane releases proapoptotic proteins such as cytochrome c, apoptosis-inducing factor, and others, that could activate caspases enzymes, which are the executors of the apoptotic process (7). In the present study, we observed that LP reperfusion prevented both MPTP opening, necrosis, and apoptosis processes. Mitochondria isolated from low-pressure hearts were more resistant to Ca2+ loading (Fig. 4). CK and LDH release as well as TTC staining (Figs. 2 and 3) showed that low pressure was also associated with less necrosis compared with NP hearts. With regard to apoptosis, our results showed an increased leakage of cytochrome c from hearts reperfused at normal pressure (Fig. 6) and also an increased activity of caspase-3 (Fig. 7), suggesting that apoptosis was significantly reduced in hearts reperfused at low pressure after prolonged cold ischemic arrest.

In conclusion, this study demonstrates that low-pressure reperfusion protects the myocardium after a prolonged hypothermic ischemia. Our results suggest that this protection may involve a reduction of oxygen free radicals production and consecutive inhibition of MPTP opening. Low-pressure (or low-flow) reperfusion, like postconditioning, proceeds after ischemia at the initiation of reperfusion. This maneuver offers a reliable alternative strategy to protect the ischemic heart, simple to apply in clinical settings.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by "La Fondation de France."


   ACKNOWLEDGMENTS
 
We are grateful to Colette Berthet, Claire Rodriguez, and Robert Rousson (Biochemistry Laboratory of Cardiovascular Hospital of Lyon) for excellent assistance and Georges Christé for editing the English.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Ferrera, EMI-U 0226, Laboratoire de Physiologie, Faculté de Médecine Lyon-Nord, 8, Ave. Rockefeller, 69373 LYON Cedex 08, France (e-mail: ferrera{at}lyon.inserm.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
 GRANTS
 REFERENCES
 

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