[ d -Ala2,d -Leu5]-enkephalin (DADLE) and morphine-induced postconditioning by inhibition of mitochondrial permeability transition pore,in human myocardium

Experimental conditions

As previously described, ²¹ one or two trabeculae were isolated from right atrial appendage, and placed vertically between an isometric force transducer (MLT0202; ADInstruments, Sydney, Australia) and maintained by clip in a 200 ml jacketed reservoir contained Tyrode's modified solution (120 mmol NaCl, 3.5 mmol KCl, 1.1 mmol MgCl₂, 1.8 mmol NaH₂PO₄, 25.7 mmol NaHCO₃, 2.0 mmol CaCl₂, and 5.5 mmol glucose, pH 7.40). The Tyrode's modified solution was insufflated with 95% O₂–5% CO₂, resulting in a partial pressure of oxygen of 600 mmHg. A thermostatic water circulator (Polystat micropros; Bioblock, Illkirch, France) maintained the temperature of reservoir at 34°C. Trabecualae were field-stimulated by two platinum electrodes at 1 Hz with rectangular wave pulses of 5-ms duration 20% above threshold (CMS 95107; Bionic Instrument, Paris, France).

A period of equilibration (90 min) was respected, permitting that trabeculae develop their mechanical performance at the apex of the length active isometric tension curve (L _max). The developed force was measured in continuous, digitized at a sampling frequency of 400 Hz and the data stored in a computer (PowerLab; ADInstruments).

At the end of experiment, the muscle cross-sectional area was calculated from its weight and length assuming a cylindrical shape and a density of 1. To avoid including sample suffering from core hypoxia, We excluded trabeculae with a cross-sectional area grater than 1.0 mm², a force of contraction normalized per cross-sectional area (FoC) less than 5.0 mN/mm², and a ratio of resting force/total force grater than 0.50 otherwise they were excluded a posteriori. ²²

Experimental protocol

At the end of the equilibration period, the trabeculae were randomized into eight groups. In all groups, trabeculae were exposed to the hypoxia reoxygenation protocol, by insufflation with 95% N₂–5% CO₂ in the buffer for 30 min, followed by a 60 min oxygenated recovery period, as previously described by our group ²¹ (Figure 1). OPEN IN VIEWER

In the control group (control; n = 6), no other treatment were administered (Figure 1).

In treatment groups, morphine 0.5 μmol (morphine; n = 6), DADLE 10 nmol (DADLE 10; n = 6), DADLE 50 nmol (DADLE 50; n = 6), DADLE 100 nmol (DADLE 100; n = 6) were administered during the first 15 min of reoxygenation (Figure 1).

In another two groups, DADLE 100 nmol and morphine 0.5 μmol were respectively administered in the presence of atractyloside 50 μmol (Sigma Aldrich, Saint Quentin Fallavier, France), the mPTP opener (morphine + atract and DADLE + atract; n = 6 in each group). Atractyloside was administered five minutes before morphine or DADLE and throughout the administration of morphine or DADLE (Figure 1).

In an additional group atractyloside 50 μmol was administered alone five minutes before reoxygenation and during the 15 first minutes of reoxygenation (atract; n = 6; Figure 1).

The morphine dose, used in the present study, was based on clinical practice and are comparable to a blood concentration of patient that received high doses of morphine intravenously. ²³ This DADLE range dose (nmol) has been used in the human myocardium in vitro, in a context of PC. ¹⁶ We have previously shown that atractyloside, used at a dose of 50 μmol, was sufficient to abolish cardioprotection without some effect in control conditions, in the same experimental model. ²¹

Statistical analysis

The endpoint of the study was the recovery of FoC at 60 min of reoxygenation (FoC₆₀, expressed as percent of baseline value). Taken account the number of comparisons, the power analysis calculated a group size of n = 6 to detect a difference of 40% in FoC₆₀ with a power of 0.8 at alpha-level of 0.05. Data are expressed as mean ± standard deviation (SD). Age, preoperative left ventricular ejection fraction (Table 1) and FoC₆₀ are compared by an univariate analysis of variance with group factor as the independent variable. If P value was inferior to 0.05 a post hoc test (Bonferroni) was performed. Within-group data were analyzed over time using analysis of variance for repeated-measures and Bonferroni post hoc analysis with group factor and time (baseline, hypoxia 5, 10, 20, 30 min and reoxygenation 5, 10, 20, 30, 40, 50 and 60 min) as covariates. All P values were two-tailed, and a P value of less than 0.05 was required to reject the null hypothesis. Statview 5 software (Deltasoft, Meylan, France) was used for perform statistical analysis.

Effect of morphine and DADLE on hypoxia reoxygenation

In the control group, after 30 min of hypoxia and 60 min reoxygenation resulted in a partial recovery of FoC₆₀ (53 ± 3% of baseline). As compared with control group, DADLE 10 (50 ± 9% of baseline; P = 0.60 versus control group) did not significantly modify FoC₆₀ (Figure 2). OPEN IN VIEWER

As compared with the control group, morphine (81 ± 9% of baseline; P < 0.001 versus control group), DADLE 50 (76 ± 11% of baseline; P < 0.001 versus control group), DADLE 100 (81 ± 4% of baseline; P < 0.001 versus control group) increased the FoC₆₀. There was no difference in the FoC₆₀ measured in the morphine, DADLE 50 and DADLE 100 groups (morphine group versus DADLE 50 group P = 0.29; morphine group versus DADLE 100 group P = 0.95 and DADLE 50 versus DADLE 100 P = 0.32) (Figure 2).

Effect of atractyloside on morphine and DADLE treatment

As shown in Figure 2, morphine-induced enhanced recovery of FoC₆₀ (81 ± 9% of baseline) was also abolished in the presence of atractyloside (morphine + atract: 57 ± 6%; P < 0.001 versus morphine group). The DADLE 100 nmol-induced enhanced recovery of FoC₆₀ (81 ± 4% of baseline) was abolished in the presence of atractyloside (DADLE + atract: 44 ± 7%; P < 0.001 versus DADLE 100 group; Figure 2).

Atractyloside alone has no significant effect on FoC₆₀, as compared with the control group (FoC₆₀: 51 ± 5% of baseline; P = 0.76 versus control group; Figure 2).

Morphine-induced postconditioning

Morphine is daily used in the management of pain during myocardial ischemia and in the postoperative period of cardiac surgery. Morphine is a non-selective opioid receptor agonist binding to all subtypes of opioid receptors (delta, mu, kappa), but with a higher affinity for mu receptors. This high affinity for mu receptors could be constitute a limitation, since, mu opioid receptors’ stimulation determined cardiovascular and respiratory adverse effects. ²⁴ It has been shown, in vivo, that morphine administered at the reperfusion, in rat (0.3 mg/kg), and in rabbit (5 mg/kg), significantly reduced myocardial infarct size. ^9,12 In isolated rat heart, subjected to 30 min of ischemia and two hours of reperfusion, administration of morphine in a range dose of 1–30 μmol in early period of reperfusion, reduced the infarct volume. ^5,7,13 Nevertheless, the doses used in these studies are far from those measured in clinical practice. ²³ In the present study, the morphine's concentration used are comparable to that measured in the serum of patients. ²³ Importantly, the present results strongly suggest that 15 min administration of morphine 0.5 μmol at the onset of reoxygenation could induced postC of human myocardium in vitro. The beneficial effect of morphine-induced PostC was observed on the recovery of FoC at the end of the reoxygenation period.

Concentration relationship of DADLE postconditioning

DADLE may be a promising synthetic molecule capable to induce hypometabolism state such as hibernation induce trigger, to improve the preservation of solid organs (heart, brain, kidney, liver) ¹⁵ and, if administered before ischemia, to protect organs against ischemic–reperfusion injury. ^25–27 DADLE is a selective delta opioid agonist, with an affinity nine-fold greater than that of morphine. It also binds to mu receptors with an affinity comparable to that of morphine. In the current study, 15 min administration of DADLE 50 and 100 nmol at the onset of reoxygenation enhanced the recovery of FoC₆₀ as compared with control group suggesting that DADLE induced PostC of human myocardium, in vitro. Interestingly, the present results showed that the cardioprotective effects of DADLE (50 and 100 nmol) induced PostC was comparable to that of morphine-induced PostC. Our results suggested that a lower dose of DADLE could be necessary to induce protection seeing its high affinity for delta receptors. Moreover, opioid side-effects, determined in particularly by mu opioid receptors’ stimulation, are dose-dependent so, we can then assume that DADLE could have fewer side-effects as compared with a non-selective opioid receptor agonist, such as morphine. Importantly, DADLE has a favorable pharmacokinetic data for its short half-life because is quickly metabolized by endopeptidases and for this reason is free from late complication. ²⁸ Bell et al. ¹⁶ showed that DADLE 10 nmol preconditioned human atrial trabeculae against hypoxia reoxygenation. In the present study, DADLE 10 nmol administered in the early reoxygenation period had no effect on the recovery of FoC₆₀, suggesting that a higher concentration of DADLE could be required to trigger pharmacological PostC. Thus, in isolated rabbit hearts, the concentration of DADLE able to induce PC, failed to induce PostC. ²⁹ Taken together, these results suggest that a higher concentration of DADLE could be required to trigger myocardial PostC as compared with the concentration required to trigger PC. We cannot rule out the hypothesis that increasing the dose of DADLE (from 10 to 50 nmol) results in activation of mu and kappa subtypes of opioid receptors in addition to the delta subtype. Thus, Zatta et al. ⁶ showed that both mu and delta opioid receptors were involved in ischemic PostC of rat heart. Moreover, Wong et al. ³⁰ showed that delta and kappa opioid receptors mediate ischemic PostC and PostC by remifentanil, in rat in vivo.

Involvement of mitochondrial permeability transition pore

Mechanisms involved in morphine- and DADLE-induced cardioprotection are poorly documented. A growing body of evidences suggests that mitochondrion is a major component of cellular cardioprotective pathways. Although the role of mitochondrial adenosine triphosphate-sensitive potassium channels in DADLE-induced PC has been demonstrated. ¹⁶ It has been shown, in isolated rat heart, that inhibition of mPTP mediated morphine-induced PostC. ^5,7 In addition, Kim et al. ⁷ suggested that this inhibition was dependent on the delta(1) opioid receptors in morphine-induced PostC. The key role of mPTP in cardioprotection in human myocardium has been established by Yellon's group, ²⁰ showing that inhibition of mPTP by cyclosporine A administered at the onset of reoxygenation protects human myocardium against lethal hypoxia–reoxygenation injury, in vitro. This suggests that, in the human heart in vitro, the mPTP is a viable target for cardioprotection. ²⁰ Nevertheless, its role in morphine and DADLE-induced cardioprotection of human heart has never been investigated. Our results showed that morphine and DADLE-induced PostC were abolished by mPTP opening (with atractyloside), at the onset of reoxygenation, suggesting that both morphine and DADLE administration in first minutes of reoxygenation prevents mPTP opening. The current study suggested that likely, in human myocardium in vitro, morphine and DADLE-induced PostC via the inhibition of mPTP in early reoxygenation period.

Several limits must be considered in the interpretation of the present results. First, the effects of anesthetic drugs, ^3,31 diseases and treatments received by the patients, before obtaining atrial appendages cannot be ruled out. On the other hand, our study included a control group equally affected by all these potentially confounding factors, and representative of population, that could have benefits from this type of cardioprotection strategy. Second, studies in isolated human right atrial myocardium must be performed at 34°C to obtain a stable isometric contraction during several hours. However, hypothermia is routinely used in cardiac surgery and mild hypothermia is a protective strategy during cardiopulmonary resuscitation. ³² Third, actually no data are available on the clinical pharmacokinetics and pharmacodynamics of DADLE. Fourth, our data strongly suggest a key role for mPTP inhibition in morphine and DADLE-induced PostC. Nevertheless, it has been reported that atractyloside not only opens mPTP but also inhibits adenosine diphosphate transport by inhibition of adenine nucleotide translocase, therefore limiting oxidative phosphorylation. ³³ As such, it would be difficult to differentiate the effects of atractyloside on mPTP opening versus loss of energy production as causative factors in attenuated protection. Our results also require qualification because the actions of morphine and DADLE with or without atractyloside pretreatment on mPTP channel activity in isolated mitochondria were not investigated. Fifth, the use of human right atrial trabeculae is easily accessible, relatively disease free and eliminates inter species differences. However, for ethical reason is not possible to obtain ventricular tissue. Bell et al. ¹⁶ have shown that atrium and ventricle have a similar copy number of δ and μ receptors, even if they have different structural and mechanical features.

In conclusion, administration of morphine and DADLE, in early reoxygenation, induced PostC of human myocardium in vitro, at least in part, through the inhibition of mPTP opening. Similar results have been reported in brain exposed to ischemia–reperfusion injury. ³⁴ Because DADLE has a short half-life, it could be proposed as protective agent against ischemia–reperfusion injury. Otherwise, further studies will be required to examine if this PostC effect of DADLE could be proposed in clinical setting.

Author contributions: All authors participated in the design, interpretation of the studies and analysis of the data, review of the manuscript and interpretation of the data; SL conducted the experiments; MF and SL wrote the manuscript. MF and SL contributed equally to this study.