Cardiovascular Research

Cardiovascular Research 2004 61(3):600-609; doi:10.1016/j.cardiores.2003.10.013
© 2004 by European Society of Cardiology

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Copyright © 2003, European Society of Cardiology

Delayed preconditioning of the human myocardium: signal transduction and clinical implications

Mahmoud Loubani, Ashraf Hassouna and Manuel Galiñanes^*

Department of Integrative Human Cardiovascular Physiology and Cardiac Surgery, University of Leicester, Glenfield Hospital, Groby Road, Leicester, LE3 9QP, UK

^* Corresponding author. Tel.: +44-116-2563032; fax: +44-116-2502449. mg50{at}le.ac.uk

Received 28 July 2003; revised 19 September 2003; accepted 7 October 2003

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Objective: Ischemic preconditioning confers cardioprotection in early and delayed phases. We investigated the delayed window of pharmacological and ischemic preconditioning in human myocardium, and the involvement of mitoK_ATP, PKC and p38MAPK. Methods: These studies were carried out using human right atrial tissue in a cell necrosis model. The tissue was obtained from patients undergoing coronary artery surgery. Results: The second window triggered by ischemia, phenylephrine or adenosine resulted in similar cardioprotection between 24 and 72 h following the intervention. Atrial tissue taken from patients with a single episode of angina between 24 and 72 h prior to surgery were already protected and preconditioning with ischemia, phenylephrine or adenosine did not add to the protection. The trigger of preconditioning with mitoK_ATP channel opener diazoxide, PKC activator PMA and p38MAPK activator anisomycin produced similar delayed protection to that of ischemia or phenylephrine. Cardioprotection was lost when mitoK_ATP channels were blocked by 5HD, PKC by chelerythrine and p38MAPK by SB203580 24 h after the trigger of preconditioning. Conclusions: Ischemic and pharmacological preconditioning induce similar delayed cardioprotection of the human heart. This second window of protection that is seen between 24 and 72 h occurs in vitro and in vivo and requires opening of mitoK_ATP channels and activation of PKC and p38MAPK.

KEYWORDS Delayed preconditioning; Human myocardium; Signal transduction

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Ischemic preconditioning is an inherent protective mechanism that, induced by brief periods of ischemia and reperfusion, protects the heart against prolonged ischemic damage [1]. This protection manifests itself in increased resistance to infarction [1–3], decreased reperfusion induced arrythmias [4,5] and contractile dysfunction [6,7], and slowing of adenosine triphosphate decline [8] during ischemia. This endogenous protective mechanism has been shown to exist in all animal species studied including man.

The cardioprotective effect of ischemic preconditioning occurs in two phases. The first is immediate and lasts for 2–4 h [9] while the second window of protection occurs at least 24 h following the initial sublethal ischemic insult and has been shown to last up to 72 h in certain species [10]. The early, or first window of protection, has been extensively investigated and there is evidence that the beneficial effect is mediated by Protein Kinase C (PKC) [11,12], p38 mitogen-activated protein kinase (p38MAPK) and ATP-sensitive potassium (K_ATP) channels [12–14]. We [15] have previously demonstrated the existence of delayed cardioprotection in the human myocardium, however the second window has not been fully characterized in man and the underlying signal transduction mechanism remains unclear.

The aims of these studies were: (i) to characterize the second window of ischemic and pharmacological preconditioning using an in vitro model of simulated ischemia and reoxygenation of human atrial myocardium, (ii) to examine whether the delayed cardioprotection is elicited in vivo by angina, and (iii) to investigate the role of mitoK_ATP channels, PKC and p38MAPK in the signal transduction mechanism of protection.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
2.1 Patient selection and experimental preparation
Experiments were performed on muscle obtained from the right atrial appendage of patients with triple vessel coronary artery disease undergoing elective coronary artery surgery in a cell necrosis model of simulated ischemia and reoxygenation. Patients were excluded if they had large atriums, history of atrial arrhythmias such as paroxysmal or permanent atrial fibrillation, atrial flutter or supraventricular arrhythmia, poor left ventricular function (ejection fractions <30%), diabetes, right ventricular failure or were taking oral hypoglycemic agents, potassium channel openers, opioid analgesia, or catecholamines. Table 1 demonstrates the clinical characteristics of the patients included in Studies 1–4. Local ethical committee approval was obtained for the harvesting technique and the studies conducted according to Declaration of Helsinki principles.

View this table: [in this window] [in a new window]   Table 1 Demographic data of all the patients included in the studies

 
The right atrial tissue was collected in oxygenated HEPES buffered solution at 4–5 °C and immediately sectioned and prepared for study. The appendage was mounted onto a ground glass plate with the epicardial surface faced down and then sliced freehand to prevent crushing artifact using surgical skin graft blades (Shwann-Morton, UK) to a thickness of between 300 and 500 µm. The muscles weighing 30–50 mg were transferred to conical flasks (25 ml Erlenmeyer flasks, Schott Glaswerk, Mainz, Germany) containing 10 ml of oxygenated buffered solution and the flasks were then placed in a shaking water bath maintained at 37 °C. Between two and eight specimens were obtained from each atrial appendage depending on the size and quality of the appendage. The incubation medium was oxygenated by a continuous flow of 95% O₂/5% CO₂ gas mixture to obtain a PO₂ between 25 and 30 kPa and a CO₂ between 6 and 6.5 kPa. The PO₂, PCO₂ and pH in the incubation medium were monitored by intermittent analyses of the solution by using an automated blood gas analyzer (model 855 Blood Gas System, Chiron Diagnostics) and the pH was kept between 7.36 and 7.45. For the induction of simulated ischemia, the medium was bubbled with 95% N₂/5% CO₂ (pH 6.80–7.00), D-glucose was replaced with 2-deoxy glucose and fetal calf serum was removed to deprive the tissue of nutrients.

2.2 Assessment of tissue injury and viability
Tissue viability was assessed by the reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) to a blue formazan product at the end of the experimental time. The absorbance of the blue formazan formed was measured on a plate reader (Benchmark, Bio-Rad Laboratories, California, USA) at 550 nm and the results expressed as mM/g wet wt [16]. In this preparation, the atrial tissue was not paced and the force developed was not measured.

Tissue injury was determined by measuring the leakage of creatine kinase (CK) into the incubation medium during the 120 min reoxygenation period. The CK measurements in the results represent the amount of CK leaked only during the 2 h of reperfusion at the end of the experimental protocol. This was assayed by a kinetic ultraviolet method based on the formation of NADH which is directly proportional to the amount of CK activity (Sigma Catalogue No. 1340-K as recommended by the German Society for Clinical Chemistry) and the results expressed as U/g wet wt.

2.3 Solutions and drugs
The incubation medium was prepared daily with deionized distilled water and contained (in mmol/l): NaCl₂ (118), KCl (4.8), NaHCO₃ (27.2), MgCl₂ (1.2), KH₂PO₄ (1.0) CaCl₂ (1.25), D-glucose (10), HEPES (20), 10% fetal calf serum and 100 µl of gentamicin per 10 ml of solution with a final concentration of 5 mg gentamicin/10 ml of incubation medium. Phenylephrine, 5-hydroxydecanoate and anisomycin were used dissolved in deionized distilled water, while adenosine, diazoxide and phorbol 12-myristate 13-acetate (PMA) were dissolved in DMSO. The final concentrations of DMSO in the incubation medium were between 1x10^–6 and 1x10^–8% and these doses were demonstrated to have no effect on the atrial tissue in our model (data not provided) in experiments prior to the start of the current studies. Anisomycin is an antibiotic that inhibits protein synthesis and has been demonstrated to activate p38MAPK while PMA is a phorbol ester that is widely used to activate PKC. All the drug doses were chosen following extensive preliminary dose–response studies. The use of 10 µM of SB203580 was chosen after carrying out dose–response experiments to identify the dose required to block the activity of p38MAPK in our preparation. However, this has been shown to offer a degree of inhibition of JNK in other preparations [17]. All reagents were obtained from Sigma.

2.4 Study protocols
All atrial muscles were allowed to equilibrate under aerobic conditions for 30 min prior to being included in a study protocol.

2.4.1 Study 1: to assess the durability and viability of the preparation
Atrial muscles (n=8/group) were aerobically perfused for various periods ranging from 0 to 480 h.

2.4.2 Study 2: to define the second window of ischemic and pharmacological preconditioning
The muscles were randomized into one of the following groups (n=8/group): (i) aerobic time-matched control, (ii) 90 min of simulated ischemia/120 min reoxygenation (SI/R), (iii) ischemic preconditioning with 5 min of ischemia and 5 min reoxygenation prior to SI/R, (iv) phenylephrine (0.1 µM) for 5 min/5 min washout prior to SI/R and (v) adenosine (100 µM) for 5 min/5 min washout prior to SI/R. Samples were aerobically perfused for varying periods of time (0, 12, 24, 48, 72 or 96 h) following the preconditioning protocol and prior to the 90 min of SI/120 min R.

2.4.3 Study 3: to study the in vivo effect of angina on preconditioning
In this study, atrial appendages were taken from 15 patients undergoing coronary artery bypass surgery who have had a single episode of angina of between 5 and 10 min duration prior to surgery. These were taken from patients who had their angina at varying times from 5 to 81 h prior surgery. All the patients were in hospital and accurate time of the episode of angina and duration were documented by medical staff. Also ECG and cardiac enzymes at the time of angina were taken to exclude individuals who suffered a myocardial infarction. The atrial appendages from each patient were then sliced and randomly assigned to one of the following groups: (i) aerobic perfusion, (ii) 90 min SI followed by 120 min R, (iii) ischemic preconditioning with 5 min ischemia/5 min reoxygenation prior to SI/R, (iv) phenylephrine (0.1 µM) for 5 min and then 5 min washout prior to SI/R, and (v) adenosine (100 µM) for 5 min and then 5 min washout prior to SI/R.

2.4.4 Study 4: to investigate the role of mitoK_ATP channels, PKC and p38MAPK in the signal transduction pathway of cardioprotection by delayed preconditioning
To achieve this, the following groups, also shown in Fig. 1, were studied (n=8/group): (i) 24 h aerobic perfusion, (ii) 90 min SI/120 min R, (iii) ischemic preconditioning with 5 min ischemia/5 min reoxygenation, (iv) phenylephrine (0.1 µM) for 5 min/washout for 5 min, (v) diazoxide (100 µM) for 10 min, (vi) PMA (1 µM) for 10 min, (vii) anisomycin (1 nM) for 10 min (viii) 5-hydroxydecanoate (100 µM) for 10 min prior to ischemic preconditioning, (ix) chelerythrine (10 µM) for 10 min prior to ischemic preconditioning, (x) SB 203508 (10 µM) for 10 min prior to ischemic preconditioning, (xi) 5-hydroxydecanoate (100 µM) for 10 min prior to preconditioning with phenylephrine, (xii) chelerytherine (10 µM) for 10 min prior to preconditioning with phenylephrine, and (xiii) SB 203508 (10 µM) for 10 min prior to preconditioning with phenylephrine. In groups (ii) to (xiii), each intervention was followed by 24 h of aerobic perfusion prior to 90 min SI/120 min R.

View larger version (34K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Schematic representation of the protocol for study 4. Eq: 30-min equilibration; SI: simulated ischemia; R: reoxygenation; P: phenylephrine; DZX: diazoxide; PMA: phorbol 12-myristate 13-acetate; ANS: anisomycin; 5HD: 5-hydroxydecanoate; CHE: chelerythrine; SB: SB203580.

 
2.5 Statistical analysis
All data are presented as mean±S.E.M. Mean values were compared by ANOVA with application of a post hoc Tukey's test. Statistical significance was taken as p<0.05.

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
All specimens that were randomized and entered the study were included in the analysis.

3.1 Study 1: durability and viability of the preparation
As shown in Fig. 2, the mean MTT values for the first 12 days were similar to those observed in fresh muscle and in the muscle aerobically incubated for only 30 min; however, MTT was significantly reduced by more than half of the fresh muscle group by 20 days of aerobic incubation. These results suggest that in this preparation the myocardial tissue remains viable for at least 12 days.

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 MTT reduction in atrial myocardium at the end of increasing periods of aerobic incubation.

 
3.2 Study 2: characterization of the second window of protection
Fig. 3A shows that SI/R resulted in a significant decrease in MTT mean values and that both ischemic and pharmacological preconditioning with phenylephrine or adenosine triggered significant early protection (0 h between preconditioning and SI/R—first window). Protection was lost 12 h after the preconditioning intervention but this was regained by 24 h, it was maintained up to 72 h and then it was lost beyond this period. A mirror image of the MTT results were observed with the CK leakage values that are shown in Fig. 3B, for the first 24 h; however, beyond this period CK release fell sharply in all study groups including the aerobically incubated group. Our laboratory has previously demonstrated [18] that in this in vitro atrial muscle preparation CK is continuously released during aerobic incubation of the tissue and that therefore measurements of CK leakage into the media may not be an appropriate index of tissue injury beyond 24 h of incubation.

View larger version (40K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 MTT reduction at the end of the reoxygenation period (A) and creatine kinase (CK) leakage into the media (B) during the 120-min reoxygenation period in human atrial myocardium subjected to various protocols (see text for details) to define the second window of ischemic and pharmacological preconditioning. Data are expressed as mean±S.E.M. of eight experiments. *p<0.05 vs. corresponding simulated ischemia/reoxygenation alone group.

 
3.3 Study 3: in vivo preconditioning by angina
Fig. 4A and B shows the individual MTT and CK leakage values of atrial muscles from patients having angina prior to surgery and subjected to various protocols of ischemic and pharmacological preconditioning. The results demonstrate that the muscles from patients presenting with angina 20 h of surgery were not preconditioned and that preconditioning with ischemia, phenylephrine or adenosine elicited similar cardioprotection. They also show that between 29 and 70 h of the episode of angina the muscles are preconditioned and that preconditioning either with ischemia or pharmacologically with phenylephrine or adenosine does not confer additional protection as reflected by the MTT and CK values. However, protection was lost beyond 70 h of the angina episode and the tissue could be preconditioned again with ischemia or pharmacologically.

View larger version (29K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 MTT reduction by the slices at the end of the reoxygenation period (A) and creatine kinase (CK) leakage into the media (B) during the 120-min reoxygenation period by human atrial myocardium subjected to various protocols (see text for details) to study the effect of angina on preconditioning). Every time point represents the muscle from one patient subjected to the various protocols.

 
3.4 Study 4: the role of mitoK_ATP channels, PKC and p38MAPK
Fig. 5A and B shows the results for the MTT reduction and the CK leakage and demonstrate that preconditioning with ischemia and pharmacologically with phenylephrine have a similar protection of the myocardial tissue at 24 h. This protective effect was matched by opening of mitoK_ATP channels with diazoxide and by activation of PKC with PMA or p38MAPK with anisomycin and blockade of each of these three factors resulted in the loss of the cardioprotective effect.

View larger version (59K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 MTT reduction at the end of the reoxygenation period (A) and creatine kinase (CK) leakage into the media (B) during the 120-min reoxygenation period by human atrial myocardium subjected to various protocols (see text for details) to investigate the signal transduction of preconditioning. Data are expressed as mean±S.E.M. of eight experiments. *p<0.05 vs. simulated ischemia/reoxygenation alone group. SI/R: simulated ischemia/reoxygenation; IP: ischemic preconditioning; 5HD: 5-hydroxydecanoate; CHE: chelerythrine; SB: SB203580.

 

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
The present studies have demonstrated that ischemic and pharmacological preconditioning equally elicit a delayed or second window of protection in the human myocardium that lasts between 24 and 72 h following the preconditioning stimulus. They have also shown that the occurrence of angina mimics the delayed protection conferred by ischemic and pharmacological preconditioning and that mitoK_ATP channels, PKC and p38MAPK are essential components of the signal transduction mechanism of this delayed protection. The clinical importance and the contribution of these results to the understanding of the mechanism underlying the delayed protection of preconditioning warrant further discussion.

4.1 The delayed phase of preconditioning
A previous report from our laboratory [15] using a similar but not identical in vitro preparation to the one used in the present studies and another study from Arstall et al. [19] using fetal cardiomyocytes have shown evidence of delayed cardioprotection in the human myocardium. Here we have now fully characterized for the first time the phenomenon of delayed protection in man and shown that this is confined to a period between 24 and 72 h following the preconditioning trigger. Our previous findings [15] showed that the beneficial effect of the second window of preconditioning was not as potent as the protection of the first window and this contrast with the present studies demonstrating that the early and delayed protection of preconditioning are equipotent as suggested by the results on MTT and CK leakage. A possible explanation for the difference between the two studies may be that in the current investigations the incubation medium was changed every 12 h and it was supplemented with fetal calf serum, which may have made the preparation more stable. Furthermore, the addition of antibiotics to the medium may have prevented the growth of bacteria and this also may have contributed to a more stable preparation. However, the controversy is further fuelled by the observation that in the infarct size model the delayed protection is less effective than the early window in the rabbit [20] and in the dog [21]. Indeed, additional studies may be required to elucidate this issue.

It is worth noting that the delayed cardioprotection elicited in the human myocardium by pharmacological preconditioning with adenosine and phenylephrine exhibited a similar potency and identical window of protection to that of preconditioning with ischemia. Experimental evidence for a role of adenosine and phenylephrine in delayed cardioprotection has been found in the rabbit [21] and in the mouse [22]. In the present studies, we only investigated the role of adenosine and phenylephrine; however, it is likely that other triggers such as reactive oxygen species [23], nitric oxide [24], bradykinin [25], opioids [26] and prostanoids [27], which have been shown to play a role in the delayed protection in animal studies, would also be operative in the human myocardium.

It should be clarified that although the benefit on the MTT results for the entire second window were similar to those seen in the first window, the CK leakage values declined when muscles were incubated for periods longer than 24 h. This pattern of CK release is probably a consequence of the constant enzyme leakage into the incubation media [18] and the resultant gradual lower tissue content. Therefore, CK values beyond the 24-h incubation period may not represent the degree of the ischemic insult to which the muscle is subjected in this preparation and they should be interpreted with caution.

4.2 Preconditioning with angina
Our finding that an episode of angina results in delayed preconditioning of the atrial myocardium against an ischemic insult, as denoted by the assessment of CK leakage and MTT reduction, is supported by the demonstration that angina preceding myocardial infarction by 24 h results in limitation of the infarct size [28] and reduction of the incidence of cardiac events [29]. It has also been reported that the administration of nitroglycerin and the occurrence of angina 24 h before percutaneous transluminal coronary angioplasty are equipotent in decreasing chest pain score during the procedure [30]. In the present studies, cardioprotection was absent when the ischemic insult was induced between 5 and 20 h of the episode of angina, it was present between 29 and 70 h after the angina and it was again lost beyond this period. In spite of the failure of angina to precondition outside this well-defined time period, the muscles maintained the potential to become protected by the acute application of ischemic preconditioning and by the administration of adenosine and phenylephrine. The results also show that once the protection is obtained by one of the preconditioning stimuli the application of additional preconditioning triggers does not lead to an increased level of protection. It has been reported, however, that the protection conferred by ischemic preconditioning may be enhanced when combined with adenosine in sheep hearts [31]. If ischemic and pharmacological preconditioning are using identical transduction pathway, it is difficult to accept that combination of the two treatments results in additional cardioprotection. Therefore the most probable explanation for the results of the latter study is that the ischemic preconditioning protocol was insufficient to elicit maximal protection and that this was only obtained when the two interventions, ischemic preconditioning and adenosine, were applied in combination.

Although all the patients had triple vessel coronary artery disease in our study, the right atrium may not have been subjected to a direct preconditioning ischemic stimulus but rather the atrial tissue may have become preconditioned at a distance by preconditioning other parts of the heart. Indeed, the concept of remote preconditioning has been demonstrated so that the whole heart can be protected by preconditioning of a selected myocardial area [32] and by distal preconditioning of other organs such as the kidneys [33,34].

4.3 Signal transduction mechanism
Our demonstration that similar cardioprotection to the one obtained with ischemic and pharmacological preconditioning can be achieved by opening the mitoK_ATP channels and by activating PKC and p38MAPK and that blockade of any of these three factors abrogates protection suggests that all three are essential components of the signal transduction pathway of the delayed or second window preconditioning in the human myocardium. A role for the mitoK_ATP channels in the delayed protection of preconditioning has also been shown in rabbits [35,36] and a participation of PKC and p38MAPK has also been suggested in the rabbit [37,38] and in the dog [39,40]. We have previously shown that the three factors are also essential components of the early or first window of preconditioning [12], thus suggesting that the signal transduction pathways of the first and second window of preconditioning may be identical in man. The brief opening of the mitoK_ATP channels with diazoxide or activation of PKC or p38MAPK triggered the entrance into both a first and then a second window of protection even though we know that the channels would have closed within minutes of the removal of diazoxide. Thus not only are the mitoK_ATP channels involved in the second window of protection but they appear to do so during the trigger phase. What remains to be explained is the cause for the loss of protection between the first and second windows of preconditioning while still maintaining the potential for preconditioning with a new ischemic or pharmacological stimulus. While the current view is that production of new proteins is the cause for the delayed protection [41,42], a possible explanation for the loss of cardioprotection between the two windows of preconditioning may be the production of some factor(s), also triggered by the preconditioning stimulus, that would counteract transiently the action of some of the components of the signal transduction pathway and this could include the end-effector(s). However, any potential mechanism for this loss of cardioprotection can be overcome by the application of a new preconditioning stimulus. Alternatively, it may be possible that mitoK_ATP channels, PKC and p38MAPK are all upstream of both a gene expression pathway and a short-term protective event. The elucidation of the cause of this temporal loss of cardioprotection has important clinical implications and will require further investigation.

	Notes

 
Time for primary review 00 days

	References

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 

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