Cardioprotection by preconditioning can be induced by various stimuli, such as brief cycles of ischemia or pharmacological agents, including volatile anesthetics and morphine1,2. Both ischemic and morphine preconditioning protect the heart by sharing common cellular pathways. Opening of mitochondrial ATP-sensitive potassium (mKATP) channels is involved in regulation of mitochondrial functions, representing a key step in mediating the cardioprotective effects of ischemic- and morphine-induced preconditioning, possibly due to inhibition of mitochondrial permeability transition pore (mPTP).3-5 Besides opening of mKATP channels, activation of the mitochondrial calcium sensitive potassium (mKCa) channel is also involved in preconditioning.6,7 Cao et al. demonstrated that activation of mKCa channels plays a crucial role in ischemic preconditioning and that cardioprotection, by activation of mKCa channels, is independent of mKATP channels and vice versa.8 In another study, the same authors demonstrated that preconditioning by activation of κ-opioid receptors is triggered by mKCa channels.9 However, the opioid, morphine, is predominantly a μ-receptor agonist and has only a low affinity for κ-opioid receptors.10,11 Furthermore, there is no κ-opioid agonist for clinical practice available. It is not known whether activation of mKCa channels is involved in morphine-induced preconditioning. The purpose of the present study was to test the hypothesis that morphine-induced preconditioning is mediated by activation of mKCa channels.

Methods

The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was performed in accordance with the requirements of the Animal Ethics Committee of the University of Duesseldorf, Duesseldorf, Germany.

Chemicals and reagents

All chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany).

Surgical preparation

Male Wistar rats were used for these studies. The rats were maintained on a 12:12 light/dark schedule (lights on at 0600 hr) with food and water provided ad libitum. The animals were anesthetized with pentobarbital 90 mg · kg−1 ip. After thoracotomy, the hearts were excised, mounted on a Langendorff system, and perfused at constant pressure (80 mmHg) with Krebs–Henseleit solution containing (in mM) 116 NaCl, 4.7 KCl, 1.1 MgSO4, 1.17 KH2PO4, 24.9 NaHCO3, 2.52 CaCl2, 8.3 glucose, and 2.2 pyruvate at 37°C. A fluid-filled balloon was inserted into the left ventricle, and end-diastolic pressure was set at 1-4 mmHg. All hearts underwent a stabilization period of 20 min. Heart rate (HR), myocardial function (isovolumetric left ventricular pressure), coronary flow, left ventricular end-diastolic pressure, and rate of left ventricular pressure development (dP/dtmax) were measured continuously. The data were digitized using an analogue to digital converter (PowerLab/8SP, ADInstruments Pty Ltd, Castle Hill, Australia) at a sampling rate of 500 Hz, and they were recorded continuously on a personal computer using Chart for Windows v5.0 (ADInstruments). Maximal contracture and time to maximal contracture were noted during ischemia. Arrhythmic intervals were not used for data analysis.

Experimental protocol for infarct size determination

The hearts were assigned randomly to one of six experimental groups (Figure 1). The hearts of all groups underwent 30 min of ischemia followed by 60 min of reperfusion. In the control group (Con), the hearts were kept under baseline conditions prior to ischemia. To investigate whether morphine induces preconditioning (MPC), morphine 1 μM was given in two five-minute cycles, separated by one five-minute washout period, and ending with one final ten-minute washout period prior to ischemia. The morphine concentration was chosen because Liang et al. demonstrated that the preconditioning effect of morphine was maximal at 1 μM.12 Morphine was dissolved in 0.9% NaCl and separately infused into a mixing chamber placed in the perfusion system. As a positive control, the ischemic preconditioning group (IPC) underwent two similar five-minute cycles of ischemia ten minutes prior to ischemia. To test whether mKCa channels are involved in the phenomenon of preconditioning, the mKCa channel inhibitor, paxilline 1 μM,8,9 was given over 25 min together with morphine- and ischemic-induced preconditioning (MPC + Pax and IPC + Pax). To rule out an effect of paxilline itself, we investigated the effect of paxilline alone (Pax).

Fig. 1
figure 1

Experimental protocol. IPC = ischemic preconditioning; MPC = morphine preconditioning; Pax = paxilline

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After 60 min of reperfusion, the heart was cut into transverse slices, which were then stained with 0.75% triphenyltetrazoliumchloride solution. The infarcted area was determined by planimetry using SigmaScan Pro 5® computer software (SPSS Science Software, Chicago, IL, USA).

Statistical analysis

The sample size was calculated using GraphPad StatMate™ Version 1.01 (GraphPad Software, San Diego, CA, USA). Sample size analysis revealed that a group size of n = 8 was necessary to detect a difference in infarct size of 25% with a power of 80% and an α < 0.05. The estimations of the mean difference of 25% and the standard deviation (SD) of 15% were based on our own data.13 Data are expressed as mean ± SD. Heart rate (in min−1) and mean aortic pressure (in mmHg) were measured during baseline, coronary artery occlusion, and reperfusion period. Comparisons of hemodynamics between groups or between time points in a group were performed (SPSS Science Software, version 12.0.1) using two-way analysis of variance followed by Dunnett’s post hoc test. A researcher blinded to the experimental groups determined the infarct sizes. The infarcts were analyzed by Student’s t test followed by Bonferroni’s correction for multiple comparisons. Changes within and among groups were considered statistically significant if P < 0.05.

Results

No differences in body or heart weight were observed between the groups (Table 1). In the preconditioning groups, the level of maximal ischemic contracture was significantly lower, and the time of maximal ischemic contracture was significantly higher compared with the Control group (P < 0.05 vs Con) (Table 1).

Table 1 Weights and ischemic contracture
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Infarct size measurement

In the Control group, infarct size was (mean ± SD) 45 ± 9% of the area at risk (Figure 2). In the ischemic- and morphine-induced preconditioning groups, infarct size was similar and significantly less than in the Control group (IPC: 20 ± 5%, MPC: 23 ± 8%; each P < 0.05 vs Con) (Figure 2). The preconditioning effect of ischemia and morphine was attenuated significantly by the mKCa-channel inhibitor, paxilline. Infarct size was 36 ± 6% and 37 ± 7% in the IPC + Pax and MPC + Pax groups, respectively; each P < 0.05 vs IPC and MPC, respectively (Figure 2). Paxilline alone had no effect on infarct size (Pax: 46 ± 7%; not significantly different from Con). There was no significant difference in infarct size between the preconditioning groups with paxilline compared with the Control group.

Fig. 2
figure 2

Infarct size measurement. Histogram showing the infarct size (IS) as percent of area at risk (AAR) in Controls (Con, n = 8), morphine preconditioning (MPC, n = 8), ischemic preconditioning (IPC, n = 8), morphine preconditioning with paxilline (MPC + Pax, n = 8), ischemic preconditioning with paxilline (IPC + Pax, n = 8), and paxilline alone (Pax, n = 8). Data are mean ± SD; * P < 0.05 vs Con; # P < 0.05 vs IPC; § P < 0.05 vs MPC

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Hemodynamic variables

Hemodynamic variables are summarized in Table 2. No significant differences in left ventricular end-diastolic pressure and dP/dtmax were observed between the experimental groups during baseline conditions and at the beginning of ischemia (Table 2). At the end of the experiment, dP/dtmax was higher in the preconditioning groups (Table 2). There was no difference in HR compared with Controls at baseline and during reperfusion, with the exception of the paxilline group at time point final ten-minute washout shortly before index ischemia (Table 2).

Table 2 Hemodynamic variables
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Discussion

The main finding of our study is that the opioid, morphine, initiates preconditioning in a similar manner as ischemia, i.e., by activation of mKCa channels.

Ischemic preconditioning (IPC) describes a cardioprotective phenomenon where short periods of myocardial ischemia protect the heart against a subsequent longer period of ischemia and reduce the deleterious consequences of ischemia/reperfusion injury.14 Besides ischemic stimuli, volatile anesthetics15,16 and morphine can mimic the cardioprotective effect of preconditioning.17 In contrast to volatile anesthetics, morphine can be administered to patients who are subjected to organ ischemia (vascular surgery, organ transplantation, cardiac surgery) or who recently underwent regional ischemia (stroke, angina pectoris, myocardial infarction, organ transplantation) without the side effect of being “anesthetized”. The mechanisms by which opioids protect the myocardium share common pathways with ischemic preconditioning. Opening of mitochondrial ATP-sensitive potassium (mKATP) channels that are involved in regulating mitochondrial functions is a key step that mediates cardioprotection induced by both morphine and ischemic preconditioning, possibly through inhibition of mitochondrial permeability transition pore (mPTP) opening.3,5

Mitochondrial calcium sensitive potassium (mKCa) channels seem to be another class of K+ channels, apart from mKATP channels, that mediate cardioprotection by preconditioning.6,7 In 2002, Xu et al.7 reported that KCa-activated potassium channels are located on the inner mitochondrial membrane and mediate cardioprotection against ischemia and reperfusion injury in the isolated perfused guinea pig heart. Since this initial observation, mKCa-activated potassium channels have been found to be implicated in ischemic preconditioning against ischemia and reperfusion injury in isolated rat and mouse hearts, anesthetized dogs, and isolated cardiac myocytes.6,8,9,18,19

The mKCa channel contains a pore forming α-subunit and a regulatory β-subunit.20-22 The β-subunit consists of four accessory β-subunits (β1-4). Both mKATP and mKCa channel activation triggers preconditioning that is independent one from the other and involves the mPTP. 8 Cao et al. showed that ischemic preconditioning is triggered by activation of mKCa channels and is abolished by the mKCa channel inhibitor, paxilline.8 Paxilline is a mycotoxin produced by the fungus, Penicillium paxilli. It has the ability to block all subunits23 of mKCa channels and is a selective inhibitor.24 Besides paxilline, there are not many mKCa channel blockers available. Iberiotoxin, for example, is a mKCa channel blocker that is also suitable for in vivo use,13 whereas paxilline is predominantly used for in vitro experiments. However, we did not check whether iberiotoxin also abolishes morphine preconditioning. Besides ischemic preconditioning, our results demonstrate that pharmacological preconditioning with morphine also induces cardioprotection by activation of mKCa channels. Cao et al.8 administered the mKCa channel blocker, paxilline, at the onset of reperfusion after a prolonged period of ischemia. We applied paxilline during the preconditioning period, suggesting that mKCa channel inhibition blocks the infarct size reducing effect of preconditioning during the trigger phase. In a recent study, we could demonstrate that activation of mKCa channels not only reduced infarct size by preconditioning but also caused a significant reduction in the mitochondrial respiratory control index.13 Co-administration of the mKCa channel blocker, iberiotoxin, completely abolished the reduction in the respiratory control index, and we concluded that cardioprotection is mediated by activation of mKCa channels leading to mild mitochondrial uncoupling. Mild mitochondrial uncoupling during the trigger phase of preconditioning may represent a common characteristic of mitochondria in a “conditioned” state.7,25-27 The involvement of mKCa channels in morphine-induced preconditioning was addressed in our study.

We demonstrated here that ischemic- and morphine-induced preconditioning reduced the infarct size to a similar extent. The mKCa channel blocker, paxilline, abolished both effects, confirming the findings of others that ischemic preconditioning involves activation of mKCa channels. Furthermore, it supports our hypothesis that mKCa channels are involved in the trigger phase of morphine-induced preconditioning. Heinen et al. showed that opening of mKCa channels can cause a slight increase in mitochondrial reactive oxygen species generation.28 The mKCa channel agonist, NS1619, requires superoxide radical generation during the preconditioning stimulus to induce a cardioprotective effect.29 Furthermore, these authors demonstrated that cardioprotection by NS1619 reduces mitochondrial calcium overload and mitochondrial reactive oxygen species production during the subsequent period of ischemia and early reperfusion.29 Such a reduction in mitochondrial calcium overload and reactive oxygen species generation has been suggested to prevent mPTP opening.30,31 Evidence suggests that the mKCa channel is located upstream of the mPTP, because cardioprotection induced by activation of mKCa channels was abolished by opening of the mPTP. Vice versa, inhibition of the mKCa channel with paxilline did not block protection induced by inhibition of the mPTP with cyclosporine A (CsA).8 Whether cardiac preconditioning by morphine is also mediated by regulation of the mPTP due to mKCa channel activation is yet unknown.

The results of the present study have to be interpreted within the scope of some limitations. First, we did not investigate possible upstream mechanisms of mKCa channels. Opioid receptors are G-protein coupled receptors whose activation inhibits adenylyl cyclase. The post-receptor signalling following opioid receptor activation has not been well defined. We cannot rule out that morphine confers preconditioning via intracellular pathways leading to activation of mKCa channels. However, the means by which morphine regulates mKCa channel activation to induce preconditioning is unknown. It has been shown that mKCa channel activation is involved in desflurane preconditioning and that protein kinase A (PKA) is located upstream of the mKCa channel.32 The activity of PKA as a possible upstream activator of KCa channels depends on the cellular level of cyclic adenosine monophosphate (cAMP)—PKA is known as cAMP-dependent protein kinase. Gross et al. demonstrated that morphine-induced cardioprotection involves glycogen synthase kinase-3beta (GSK3beta) and Akt (also called PKB).33 However, we did not investigate in the present study whether these enzymes are related to mKCa channels.

Another limitation of our study is that we did not determine the effect of ischemic and morphine preconditioning on the mitochondria. Xi et al. could show that morphine prevents mPTP opening by inactivation of GSK3beta.34 Our results showing that morphine confers preconditioning through activation of mKCa channels and the fact that enzymes like PKA regulate mKCa channel activation suggest future directions for investigating the underlying mechanism of morphine-induced cardioprotection. Clinically, morphine might be administered to patients who are subjected to organ ischemia (vascular surgery, organ transplantation, cardiac surgery) or who recently underwent regional ischemia (stroke, angina pectoris, myocardial infarction, organ transplantation) without the side-effect of being “anesthetized”. Furthermore, there is no κ-opioid agonist for clinical practice available. Therefore, unravelling the exact mechanisms of morphine-induced cardioprotection might have clinical consequences.

In summary, our results demonstrate that, besides ischemic preconditioning, morphine also initiates cardiac preconditioning via activation of mKCa channels.