Myocardial Preconditioning Against Ischemia-Induced Apoptosis and Necrosis in Man

Journal of Surgical Research 134, 138 –144 (2006) doi:10.1016/j.jss.2005.12.009

Myocardial Preconditioning Against Ischemia-Induced Apoptosis and Necrosis in Man Hunaid A. Vohra, MRCS, M.D., and Manuel Galiñanes, M.D., Ph.D., FRCS1 Cardiac Surgery Unit, Department of Cardiovascular Sciences, University of Leicester, Leicester, United Kingdom Submitted for publication October 19, 2005

Background. Ischemic preconditioning (IPC) protects against apoptosis and necrosis but the contribution of the two forms of cell death and whether the beneficial effects are mediated by similar or different signal transduction pathways remains unclear. Here we have investigated the effect of IPC on the type of cell death in the human heart and whether the inhibition of apoptosis and necrosis by IPC requires the opening of mitoK ATP channels and the activation of PKC and p38MAPK. Materials and methods and results. Free-hand tissue sections (n ⴝ 6/group) obtained from the right atrium of patients at the time of coronary bypass surgery were subjected to 90-min simulated ischemia followed by 120min reoxygenation (SI/R) with or without IPC (5 min SI/5 min R) prior to SI/R. IPC reduced apoptosis from 30.0 ⴞ 3.8 to 11.0 ⴞ 1.5% (P < 0.05) by TUNEL technique and necrosis from 11.6 ⴞ 2.4 to 4.2 ⴞ 1.7% (P < 0.05) by propidium iodide staining. When inhibitors of mitoK ATP channels (1 mM 5-hydroxydecanoate), PKC (10 ␮M chelerythrine), and p38MAPK (10 ␮M SB203580) were added for 10 min before SI, the protection against necrosis was abolished. However, whereas 5-hydroxydecanoate and chelerythrine also abolished the protection of IPC against apoptosis, SB203580 did not. The activation of mitoK ATP channels (100 ␮M diazoxide), PKC (1 ␮M PMA), and p38MAPK (1 nM anisomycin) were a mirror image of the findings with blockers. Conclusions. IPC protects the human myocardium against both apoptosis and necrosis. The anti-necrotic effect is mediated by the opening of mitoK ATP channels and activation of PKC and p38MAPK; however, the anti-apoptotic effect requires opening of the mi-

1

To whom correspondence and reprint requests should be addressed at Cardiac Surgery Unit, Department of Cardiovascular Sciences, University of Leicester, Glenfield Hospital, Groby Road, Leicester LE3 9QP, United Kingdom. E-mail: [email protected].

0022-4804/06 $32.00 © 2006 Elsevier Inc. All rights reserved.

toKATP channels and PKC activation but is p38MAPK-independent. © 2006 Elsevier Inc. All rights reserved. Key Words: apoptosis; necrosis; preconditioning; ischemia; signal transduction; man; mitochondrial KATP channels; protein kinase C; mitogen activated protein kinase; heart.

INTRODUCTION

Apoptosis and necrosis are two distinct forms of cell death. Both have been shown to be important in reperfusion injury [1]. Preconditioning by a short ischemic insult (IPC) or pharmacologically has been demonstrated to result in strong cardioprotection [2]. A variety of endogenous substances have been reported to be involved in IPC. These include mediators such as PKC and mitoK ATP channels that have been recognized in man and animals [3, 4]. The reported experimental work has usually focused on the mechanisms of preconditioning against necrosis [2, 5, 6]. However, apoptosis, like necrosis, may independently contribute to irreversible myocardial damage [7]. At present, there is no clear understanding of the mechanistic linkage of apoptotic cell death to the phenomenon of preconditioning in the human myocardium. Although both forms of cell death are thought to be closely connected to altered mitochondrial function [8, 9], it is uncertain whether the pathways to apoptosis and necrosis may share parts of the same signal transduction. The aims of the present studies performed in the human myocardium were as follows: (1) to investigate the effect of IPC on the apoptosis and necrosis induced by ischemia/reoxygenation (SI/R); and (2) to elucidate the role of mitoK ATP channels, protein kinase C (PKC), and p38 mitogen-activated protein kinase (p38MAPK).

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MATERIALS AND METHODS

Assessment of Tissue Injury

Patients

The release of creatine kinase (CK) release into the perfusate during the 2 h of reoxygenation was measured as an index of tissue injury. The enzyme activity was measured by a linked-enzyme kinetic assay employing a commercial assay kit (DG147-K: Sigma Chemicals) and expressed as IU/g wet weight.

The right atrial appendage from patients undergoing elective coronary artery bypass graft surgery was retrieved at the time of the right atrial cannulation. Patients with atrial fibrillation, poor ejection fraction (EF ⬍ 30%), and those with diabetes controlled with insulin, glyburide, glicazide, glibenclamide, or any other antidiabetic drugs, were excluded from the study. In addition, patients taking potassium channel openers (for example, nicorandil or diazoxide) [10] were also excluded. Local ethical approval and patients’ informed consent was obtained (Leicestershire Research Ethics Committee reference no. 7805). The investigation conformed with the principles outlined in the Declaration of Helsinki [11].

Experimental Preparation and Solutions The sectioning of the atrial muscle and the preparation of SI/R have been previously described [12]. Ischemia was simulated by bubbling the media with 95% N 2 and 5% CO 2 in the absence of glucose (pH 6.6 – 6.9) at 37°C. During ischemia, the pO 2 in the medium was maintained at 0 kPa, when monitored with an oxygen detector electrode (Oxylite™; Optronix Ltd, Oxford, U.K.). A pO 2 of 0 kPa is achieved within the first 10 min of the initiation of N 2 bubbling and ischemia was introduced only after this time elapsed so that the media were free of oxygen from the start of ischemia [12]. During equilibration and reoxygenation the pO 2 was maintained between 25 and 30 kPa [12]. The atrial sections were equilibrated for 30 min in oxygenated Krebs Henseleit HEPES (KHH) buffer containing (in mM): NaCl (118), KCl (4.8), NaHCO 3 (27.2) , MgCl 2 (1.2), KH 2PO 4 (1.0), CaCl 2 (1.20), glucose.H 2O (10), and HEPES (20) at a pH of 7.4 and a temperature of 37°C. The buffer was supplemented with 10% fetal calf serum (FCS: Harlansera Labs (Loughborough, England) #S-0001A). This was followed by the experimental protocols. The agents used in the present studies were purchased from Sigma Chemicals (Perth, Australia) and their doses were obtained from previous dose–response studies in our laboratory.

Experimental Protocols

Assessment of Apoptosis and Necrosis At the end of each protocol, the muscles were incubated for 10 min on ice with 5 ␮M propidium iodide (PI) in 0.1M tri-sodium citrate and 20 mM PBS at pH 7.4 to identify the necrotic nuclei. Sections were then fixed twice, first for 30 min and then with 4% paraformaldehyde in 30% sucrose and 20 mM PBS overnight on ice and at pH 7.4. Following this, serial sections of 10 ␮m were cut with a Bright cryomicrotome (model OTF) at ⫺25°C in tissue embedding matrix (Tissue Tek® OCT compound). The cryopreserved tissue sections were washed with 20 mM PBS at pH 7.4 for 2 min, then permeabilized in 0.02 mg/ml proteinase-K for 10 min at 37°C, and presensitized for 1 min in a microwave oven at 800 W in 0.1% Triton X-100 and 0.1M sodium citrate at pH 6.0. To assess apoptosis, the terminal deoxynucleotidyl transferase (TdT) was used to incorporate fluorescein (FITC)-labeled dUTP oligonucleotides to DNA strand breaks at the 3=-OH termini in a template-dependent manner (TUNEL technique) using a commercially available kit (Roche: 1684795, Basel, Switzerland). The FITC fluorescence emission (range 600 – 630 nm) was measured using argon-ion fluorescence excitation at 488 nm and detected by laser confocal epifluorescence microscopy with a ⫻10 oil immersion objective. The PI-labeled nuclei was excited with heliumneon laser light at 543 nm and fluorescence was detected using an emission range of 680 –730 nm to abolish fluorescence “bleedthrough” from FITC-labeled nuclei. Analysis was done using NIH Image software (Scion Corp., Frederick, MD) with the Cavalieri-3 macro (G. MacDonald, University of Washington). To avoid the inclusion of artefact, only fluorescent signals with areas greater than 16 ␮m2 were counted. Absolute numbers of green fluorescent apoptotic (A) and necrotic (N) red fluorescent nuclei in any given image field were determined by dividing the total number of PI-labeled nuclei (M) in the next serial, or mirror section. The absolute percentage of apoptotic cells was given by A/M*100% and the percentage of necrotic cells by N/M*100%.

Study 1: The Effect of Ischemic Preconditioning on Apoptosis and Necrosis The atrial sections (n ⫽ 6/group) were allowed to equilibrate in oxygenated KHH buffer for 30 min and then they were randomly allocated to the following protocols: (1) aerobic incubation for 210 min; (2) 90 min simulated ischemia (SI); (3) 90 min simulated ischemia followed by 120 min reoxygenation (SI/R); (4) IPC with 5 min SI plus 5 min reoxygenation followed by SI/R.

Study 2: The Role of MitoK ATP Channels, PKC, and p38MAPK on the Anti-Apoptotic and Anti-Necrotic Effects of Ischemic Preconditioning Sections of atrial muscle (n ⫽ 6/group) were equilibrated for 30 min and then subjected to the following protocols: (1) aerobic perfusion for 210 min; (2) 90 min of SI and then 120 min of R; (3) IPC prior to SI/R. In study 2A, 5-hydroxydecanoate, chelerythrine, and SB203580 were added for the last 10 min of the equilibration period and before the induction of ischemia in the SI/R and IPC groups. In study 2B, diazoxide, anisomycin, and PMA were added for the last 10 min of the equilibration period followed by a 5-min washout before the induction of ischemia.

Statistical Analysis Data were expressed as mean ⫾ standard error of the mean (SEM). Analysis of variance (ANOVA) was used for comparisons of means (Microsoft® Excel analysis tool pak) with the application of a post-hoc Tukey’s test. A P value of less than 0.05 was considered statistically significant.

RESULTS Study 1: The Effect of Ischemic Preconditioning on Apoptosis and Necrosis

CK Release As shown in Fig. 1, SI/R resulted in a significant increase in CK release when compared to aerobic perfusion and IPC significantly reduced CK leakage to mean values that were similar to those seen in the aerobic control group.

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Apoptosis and Necrosis

CK release (IU/gr wet wt)

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Fig. 4A shows that SI/R causes a significant increase in both apoptosis and necrosis when compared to the aerobic control group. Again, the increase in the two forms of cell death was unaffected by the presence of 5-hydroxydecanoate, chelerythrine, and SB203580. Interestingly, while the anti-necrotic effect of IPC was abolished by the addition of 5-hydroxydecanoate, chelerythrine, and SB203580, the anti-apoptotic effect was abolished only by 5-hydroxydecanoate and chelerythrine but not by SB203580. Fig. 4B shows that the reduction in necrosis by IPC was mimicked by the addition of diazoxide, anisomycin, and PMA; however, only diazoxide and PMA, but not anisomycin, resulted in the inhibition of apoptosis. These results were a mirror image of the results seen in study 2A.

IPC

FIG. 1. CK release into the incubation medium during 2 h reoxygenation period of right atrial muscles subjected to simulated ischemia/reoxygenation (SI/R) and ischemic preconditioning (IPC). The aerobic control served as time-matched controls. The columns represent the mean of six experiments and the bars represent the SEM. *P ⬍ 0.05 versus SI/R group.

DISCUSSION

The present studies have demonstrated that IPC protects the human myocardium against both apoptosis and necrosis and that the anti-necrotic effect is mediated by the opening of the mitoK ATP channels and activation of PKC and p38MAPK, whereas the antiapoptotic effect requires opening of the mitoK ATP channels and PKC activation but is p38MAPK independent.

Apoptosis and Necrosis

Study 2: The Role of MitoK ATP Channels, PKC, and p38MAPK on the Anti-Apoptotic and Anti-Necrotic Effects of Ischemic Preconditioning

CK Release As shown in Fig. 3A, SI/R resulted in a significant increase in CK release, which was not modified by the presence of the inhibitors 5-hydroxydecanoate, chelerythrine, and SB203580. As seen in study 1, IPC significantly reduced CK leakage to mean values that were similar to those observed in the aerobic control group. The protection of IPC was abolished by 5-hydroxydecanoate, chelerythrine, and SB203580. Fig. 3B shows that diazoxide, anisomycin, and PMA resulted in a significant reduction of CK release as compared to SI/R, and similar to the values seen with IPC.

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Fig. 2 shows that, when compared to aerobic perfusion, SI resulted in small increases in the levels of both apoptosis and necrosis that were not statistically significant. However, SI/R caused significant increases in both apoptosis and necrosis but values were greater in the former (29.5 ⫾ 2.9%) than in the latter (12.6 ⫾ 1.6%; P ⬍ 0.05) and, as expected, IPC resulted in significant reduction of both apoptosis (14.2 ⫾ 3.0%) and necrosis (7.9 ⫾ 0.9%; P ⬍ 0.05).

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aerobic control

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FIG. 2. Percentage of nuclei exhibiting apoptosis (opened columns) and necrosis (filled columns) in right atrial muscles subjected to simulated ischemia (SI), simulated ischemia/reoxygenation (SI/R), and ischemic preconditioning (IPC). The columns represent the mean of six experiments and the bars represent the SEM. *P ⬍ 0.05 versus SI/R group; †P ⬍ 0.05 versus necrosis in corresponding group.

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CK release (IU/gr wet wt)

5

sis and necrosis depends on the degree of ischemic injury and on the duration of reoxygenation. The present studies are however the first demonstrating that, in the human myocardium, the cardioprotection of IPC is mediated by a reduction of both apoptosis and necrosis. These results in the human myocardium are supported by experimental animal studies also showing that IPC reduces apoptosis [14, 15] and necrosis [2, 5, 6].

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FIG. 3. Effect of blockade (A) and activation (B) of mitoK ATP channels, PKC, and p38MAPK on the CK release during 2 h reoxygenation period of right atrial muscles subjected to simulated ischemia/reoxygenation (SI/R) and ischemic preconditioning (IPC). The columns represent the mean of six experiments and the bars represent the SEM. †P ⬍ 0.05 versus SI/R group. Abbreviations: 5-HD, 5-hydroxydecanoate; Chel, chelerythrine; SB, SB203580; DZX, diazoxide; ANS, anisomycin.

These results may contribute to a better understanding of the pathophysiology of ischemia/reoxygenation injury and cardioprotection and they are discussed below. Cardioprotection of IPC Against Apoptosis and Necrosis

Our findings that apoptosis can be a more important mechanism of cell death than necrosis after ischemia/ reoxygenation is not surprising since recent studies from our laboratory, also in the human myocardium [13], have demonstrated that the importance of apopto-

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FIG. 4. Effect of blockade (A) and activation (B) of mitoK ATP channels, PKC, and p38MAPK on the percentage of apoptosis (opened columns) and necrosis (filled columns) in right atrial muscles subjected to simulated ischemia/reoxygenation (SI/R) and ischemic preconditioning (IPC). The columns represent the mean of six experiments and the bars represent the SEM. †P ⬍ 0.05 versus corresponding IPC alone group in (A); †P ⬍ 0.05 versus corresponding SI/R group in (A); *P ⬍ 0.05 versus corresponding SI/R group in (B). Abbreviations: 5-HD, 5-hydroxydecanoate; Chel, chelerythrine; SB, SB203580; DZX, diazoxide; ANS, anisomycin.

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MitoK ATP Channels and IPC

Animal studies in the rat [15], dog [16], and also in the rabbit [17] and chick embryo cardiomyocytes [18] have previously investigated the role of mitoK ATP channels in preconditioning against necrosis but not apoptosis. The present studies have demonstrated that, in the human myocardium, mitoK ATP channels play a key role in reducing apoptosis and necrosis since cardioprotection against the two forms of cell death was lost by blockade of the channels with 5-hydroxydecanoate and elicited by opening of the channels with diazoxide. Recently we have shown that mitoK ATP channels, PKC and p38MAPK, in that order, are an integral part of the signal transduction mechanism of cardioprotection by ischemic and pharmacological preconditioning of the human myocardium [19]; however, the mechanism by which opening of mitoK ATP channels results in cardioprotection remains unclear. We [20] and other investigators [8, 21, 22] have observed that opening of mitoK ATP channels by diazoxide and nicorandil causes mitochondrial membrane potential depolarization and generation of free radicals that in turn could activate PKC (see below). An alternative mechanism of mitoK ATP channel-induced protection may be the prevention of Ca2⫹ overload seen in rat cardiac mitochondria [23]. Akao et al. [8] have demonstrated in neonatal cultured rat cardiomyocytes that opening of mitoK ATP channels by diazoxide suppresses caspase activation, the translocation of cytochrome c, and the release of poly-ADP-ribosepolymerase (PARP) caused by oxidative stress and that these effects are abolished by 5-hydroxydecanoate. However, these actions directly linked to apoptosis may represent the end-effect of the signal transduction pathway initiated by the opening of the mitoK ATP channels. PKC and IPC

The present results also provide evidence that PKC activation plays a crucial mechanistic role in IPC of the human myocardium by reducing apoptosis and necrosis. There is agreement in the literature that PKC activation reduces necrosis in in vivo and in in vitro animal studies [24, 25] but the results on the role of PKC activation in apoptosis do not agree. Thus, for example, the reported attenuation of apoptosis in rabbit and chick cardiomyocytes by PKC activation [26, 27] contrasts with the suggestion that the increased activity of the enzyme induces apoptosis in the salivary epithelium [28]. It is possible that these opposed results may find an explanation in differences in experimental conditions and the type of tissue investigated. The mechanism leading to PKC activation, the type of PKC isoform involved, and the end-effector(s) phosphorylated by PKC and responsible for the cardioprotection are not yet fully elucidated. As discussed earlier, oxygen-free radicals are strong candidates linking

the opening of mitoK ATP channels and the activation of PKC, a thesis supported by the suggestion that the oxygen-free radicals released by the mitochondria during brief episodes of hypoxia induce preconditioning in chick cardiomyocytes [9] and by the demonstration that PKC can be activated by selective oxidative modification of the regulatory domain [29]. The PKC isoforms involved is also unclear. We have shown in the human myocardium that PKC␧ is upstream and PKC␣ is downstream of the mitoK ATP channels [30]. In contrast, other investigators have observed that opening of mitoK ATP channels activates the PKC␧ isoform through oxygen-free radicals originating in the mitochondria of chick embryonic ventricular myocytes [27]. Regarding the effect of PKC activation, it can be speculated from the literature that the anti-necrotic effect of PKC activation may be mediated by regulation of Ca2⫹ channels [31], by a decrease in intracellular acidification [32], or by another as yet unknown mechanism, while its anti-apoptotic action may be related to the phosphorylation of proteins such as Bcl-2 and Bcl-X [33]. It is clear that there is scarce knowledge in this area and that more research is needed. p38MAPK and IPC

Our finding that p38MAPK plays a role in the cardioprotection of preconditioning in the human myocardium is consistent with previous studies also from our laboratory showing that p38MAPK is placed downstream of mitoK ATP channels and PKC [19]. However, in the present studies we have shown for the first time that, while the anti-necrotic effect of IPC is p38MAPKdependent, the anti-apoptotic action is p38MAPKindependent, implying that the two forms of cell death may use separate end-effectors. In the literature, the role of p38MAPK in IPC is controversial. We, here and in previous studies using human myocardium [19], and other investigators using experimental animal models such as the rabbit [34], have shown that p38MAPK activation plays a role in preconditioning, while no effect of p38MAPK activation on IPC has been seen in studies in the pig [35]. The controversy is further fueled by the observation that inhibition of p38MAPK, as opposed to activation, can be cardioprotective in the isolated rat heart [36] and rabbit heart [37]. It is possible that species differences and the experimental preparation used may provide an explanation, at least in part, for the disparate results. Tissue specificity and the temporal activation of p38MAPK have also been suggested as potential causes for the variable findings [38]. Although the role of p38MAPK in ischemic injury and in IPC is not defined, one may be tempted to speculate that any protective action is mediated by the phosphorylation of small heat shock proteins such as HSP27 via the MAPKAP kinase 2 [39], a class of proteins that has been shown to act as chaperonin of Bax

VOHRA AND GALIÑANES: PRECONDITIONING IN HUMAN MYOCARDIUM

and Bcl-X to sequester their translocation during staurosporine-induced apoptosis [40]. It should be emphasized that in many of the reported studies, including the present one, the role of p38MAPK was investigated using blockers such as SB203580, that is highly specific for the ␣- and ␤-isoforms of p38MAPK [41] but insensitive to p38␥ and p38␦ isoforms [42, 43], and that activators such as anisomycin are nonselective agents. Therefore, if p38MAPK isoforms have different physiological roles, it is possible that some of the above controversies can find an explanation on the selectivity of the p38MAPK blockers and activators used. Once again, more research is needed in this area. Clinical Implications and Study Limitations

The realization that the cardioprotective mechanism against apoptotic and necrotic cell death is in part identical but that subsequently diverges into two distinctive pathways may imply that there are two different sets of end-effectors antagonizing each of the two conditions. This opens a window of opportunity from a therapeutic point of view if apoptosis and necrosis can be specifically counteracted and if the unwanted activation of other processes can be avoided. In the present studies the human atrial tissue was used and therefore any extrapolation to the ventricular myocardium should be made with caution. However, we have shown in our laboratory (unpublished data) that a comparable level of cardioprotection occurs with preconditioning in both tissues. ACKNOWLEDGMENTS

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This work was partially supported by a BUPA Surgical Research Fellowship (H.A.V.) and a grant from Diabetes United Kingdom (RD01/0002329). We appreciate the secretarial assistance of Nicola Harris and the assistance of Dr. A. Fowler.

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