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Various models of cardiac conditioning in single or sequential periods of ischemia: Comparative effects on infarct size and intracellular signaling
International Journal of Cardiology 168 (2013) 1336–1341
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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Various models of cardiac conditioning in single or sequential periods of ischemia: Comparative effects on infarct size and intracellular signaling Konstantinos Iliodromitis a, Dimitrios Farmakis a, b, Ioanna Andreadou c,⁎, Anastasia Zoga a, Sofia-Iris Bibli c, Theodora Manolaki a, Nikolaos Dagres a, Efstathios K. Iliodromitis a, Maria Anastasiou-Nana a, Dimitrios Th. Kremastinos a a b c
Second Department of Cardiology, University of Athens Medical School, Attikon University Hospital, Athens, Greece First Department of Internal Medicine, University of Athens Medical School, Laiko Hospital, Athens, Greece Department of Pharmaceutical Chemistry, University of Athens School of Pharmacy, Athens, Greece
a r t i c l e
i n f o
Article history: Received 14 March 2012 Received in revised form 3 September 2012 Accepted 5 December 2012 Available online 25 December 2012 Keywords: Ischemia Reperfusion Infarct size Preconditioning Postconditioning Intracellular signaling
a b s t r a c t Background: Preconditioning (PreC) and postconditioning (PostC) reduce infarct size. We sought to determine the effects of PreC and PostC, alone or in combination, on infarct size and expression of intracellular signals in different ischemia models. Methods: Male rabbits were subjected to myocardial ischemia followed by 3-hour reperfusion. In a first series we applied 3 ischemia models [a 20-min period (20), a 40-min period (40), and two sequential 20-min periods (20–20)] and 3 types of interventions [no intervention (controls, C), 2 cycles of 5-min ischemia/10-min reperfusion before index ischemia (PreC) and 6 cycles of 10-s ischemia/10-s reperfusion after index ischemia and/or between the sequential ischemic periods (PostC)] (12 groups in total). Infarct size (I) and area at risk (R) were assessed (%I/R). In a second series, samples were taken for western blot analysis of Akt phosphorylation. Results: Overall, %I/R differed significantly among groups (p b 0.001). In control groups, C-40 had a greater %I/R than C-20 (p = 0.006). In intervention groups, no differences were found in %I/R. All intervention groups had significantly lower %I/R compared to C-40 group (p b 0.001), whereas, compared to C-20–20 group, PreC-20–20, 20-PostC-20, 20-PostC-20-PostC and PreC-20–20-PostC groups had lower %I/R (all p b 0.05). Akt was increased in all groups in which a significant %I/R reduction was achieved (p b 0.05 versus all other groups). Conclusions: PreC and PostC, alone or in combination, are effective when an ischemic insult of a given duration is applied either as a single or as sequential periods. Protection from either intervention is associated with an enhanced Akt activation. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Apart from the mechanical or pharmacological means of reperfusion that substantially limit myocardial necrosis during an evolving myocardial infarction, additional reduction of the final infarct size is feasible by conditioning the heart. Both preconditioning and postconditioning, local [1,2] or remote [3,4], have been extensively investigated and proved effective in preventing the reperfusion injury in all species studied so far, independently of the presence of collaterals in coronary circulation [5]. The initial experimental studies focused on the natural history of preconditioning and postconditioning [1,2,6], while later studies ⁎ Corresponding author at: Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens, Panepistimiopolis, Zografou, Athens 15771, Greece. Tel.: +30 210 7274827; fax: +30 210 7274747. E-mail address: [email protected] (I. Andreadou). 0167-5273/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijcard.2012.12.014
investigated the ligands and the intracellular transduction pathways in an attempt to substitute the mechanical means of conditioning with pharmacological agents [7–10]. The activation of the PI3K–Akt kinase pathway component of the Reperfusion Injury Salvage Kinase (RISK) pathway [11] at the onset of myocardial reperfusion has been implicated as a mediator of direct myocardial preconditioning [12] and postconditioning [13]. The acquired experience from the experimental studies has been successfully transferred into the clinical practice and various external interventions or pharmacological agents can successfully mimic conditioning and provide cardioprotection [14–18]. Following an initial ischemic insult resulting from coronary artery occlusion, a number of patients may suffer a second episode of sustained ischemia, either after a spontaneous or a therapeutic restoration of the flow. The role of pre- and postconditioning is unknown in these circumstances. Very few studies appear in the literature with two sequential periods of ischemia and to the best of our knowledge
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2.5. Western blot analysis
there is no study to test the effectiveness of these interventions when applied before, in-between and after the episodes of sustained ischemia with a parallel investigation of the intracellular mediators. In the present study, keeping in mind that several patients have a second episode of total coronary artery occlusion after an initially successful opening, we used an in vivo model of two separate and sequential periods of ischemia and reperfusion and we tested the effects of preconditioning and postconditioning on the infarct size and the RISK pathway after a single or two sequential episodes of ischemia and reperfusion.
In the second series of experiments, 48 animals were subjected to the above interventions (n = 4/group) up to the 5th min of reperfusion, when the hearts were quickly excised, immediately immersed into liquid nitrogen and then kept in −70 °C up to the time of Akt assessment. The phosphorylation state (phospho-Akt, Ser 473) and total levels of Akt were analyzed by SDS-PAGE immunoelectrophoresis using antibodies (Cell Signaling Technology) as previously described [20]. Relative densitometry was determined using a computerized software package (NIH Image) and the values for phospho-Akt were normalized to the values for total Akt.
2. Materials and methods
2.6. Statistical analysis
2.1. Animals
All results are presented as mean ± standard error of the mean. Variables were compared among the 12 groups using One-way Analysis of Variance (ANOVA) with Bonferroni correction and with Tukey post-hoc analysis. A p value b0.05 was considered as statistically significant.
A total of 143 New Zealand white male rabbits with a weight between 2.6 and 3.5 kg received proper care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institute of Health. Fourteen rabbits from different groups were excluded for various hemodynamic or technical reasons (severe pump failure during ischemia, intractable ventricular tachycardia/fibrillation, severe bleeding and infarction detected outside the area at risk). As a result, 129 animals completed the study. 2.2. Surgical procedures Sodium thiopentone at a dose of 30 mg/kg was injected into an ear vein for anesthesia. The animals were then intubated and connected to a respirator for small animals (MD Industries, Mobile, AL, USA) for mechanical ventilation at a rate adjusted to maintain normal blood gases. The experimental conditions have been previously described in detail [9,19]. A bipolar chest lead was used for continuous electrocardiographic monitoring. The chest was surgically opened with a left thoracotomy and the beating heart was exposed. The pericardium was opened and a 3-0 silk suture was passed around a prominent coronary artery. Ischemia was induced by pulling the thread through a small piece of soft tubing, which was firmly positioned against the coronary arterial wall with the aid of a small clamp. Ischemia resulted in ST elevation on the ECG and a change in the color of the myocardium. At the end of ischemic period, the snare was opened, the artery refilled and the myocardium reperfused [9,19]. 2.3. Experimental protocol The rabbits were randomly divided into 12 groups and subjected to regional myocardial ischemia followed by reperfusion. There were 3 models of ischemia [a 20-min period (20), a 40-min period (40), and two sequential 20-min periods separated by 12-min reperfusion (20–20)] and 3 types of intervention [(no intervention, controls, C), preconditioning with 2 cycles of 5-min ischemia/10-min reperfusion before index ischemia (PreC) and postconditioning with 6 cycles of 10-s ischemia/ 10-s reperfusion immediately after the index ischemia (PostC)]. The 12-min duration of the reperfusion interval was selected in order to allow comparison with the groups of preconditioning. In preconditioning, 10 min of reperfusion separates the last protective intervention of short ischemia/reperfusion from the index ischemia. Postconditioning with 6 cycles of 10-s ischemia/reperfusion account for an elapsed time of 2 min after the end of the first index ischemia. In order for all the groups to be comparable, 10 min of reperfusion was also required after the end of the last protective intervention of postconditioning and therefore a total time of 12 min was selected for the interval between the first and the second episode of sustained ischemia. The protocol is presented schematically in Fig. 1. 2.4. Risk zone and infarcted zone measurement In the first series of experiments, 81 rabbits (n = 6–8/group) followed the above protocol up to 3 h of reperfusion. At the end of reperfusion, hearts were excised, mounted on an apparatus and perfused with normal saline for 2 min for blood removal. Then the coronary ligature was retightened at the same site and 5 ml of green fluorescent microspheres were infused for the separation of the normally perfused area from the area at risk. Hearts were kept in the refrigerator for 24 h and cut into 3 mm thick sections. The slices were stained with triphenyltetrazolium chloride (TTC) at 37 °C and immersed in formaldehyde. With a wavelength of 366 nm UV light, we separated the risk from the infarcted zone of the heart and traced all the areas onto an acetate sheet in order to estimate the volumes of these areas in cm3. The tracings were subsequently scanned with the Adobe Photoshop 6.0 and measured with the Scion Image program. The areas of myocardial tissue at risk and infracted were automatically transformed into volumes. Infarct and risk area volumes were expressed in cm3 and the percent of infarct to risk area ratio (I/R) was calculated [9]. The evaluation of infarct size was performed by a researcher blinded to treatment assignment.
3. Results 3.1. Baseline characteristics Heart weight, blood pressure and heart rate of the different study groups are presented in Table 1. No significant differences were observed among the groups. 3.2. Infarct size Overall, I/R differed significantly among groups (pb 0.001). Post-hoc analyses in controls showed that C-40 group had greater I/R compared to C-20 group (43.2 ± 4.1% vs 24.5± 2.7%, p = 0.006), but not compared to C-20–20 group (34.6± 3.9%). Furthermore, I/R did not differ among the different intervention groups (13.7 ±1.2% in PreC-20, 20.6± 2.2% in 20-PostC, 19.9 ± 2.2% in PreC-40, 26.9 ± 2.4% in 40-PostC, 12.7 ± 3.5% in PreC-20–20, 20.1 ± 4.3% in 20–20-PostC, 15.7± 1.9% in 20-PostC-20 and 19.4 ±1.7% in Prec-20–20-PostC). When intervention and control groups were compared, all intervention groups had significantly lower I/R compared to C-40 group (pb 0.001), no intervention groups differed significantly compared to C-20 group, whereas compared to C-20–20 group, PreC-20–20, 20-PostC-20, 20-PostC-20-PostC and PreC-20–20-PostC groups had significantly lower I/R (p= 0.001, b0.001, 0.003, 0.011 and 0.036, respectively). The results are presented schematically in Fig. 2. 3.3. Western blot analysis The phosphorylation levels of Akt were significantly increased in all groups in which a significant I/R reduction was accomplished (Fig. 3, p b 0.05 versus all other groups). 4. Discussion The present study showed that both preconditioning and postconditioning, either alone or in combination, were effective in limiting the infarct size caused by either single or sequential periods of ischemia with a total duration of 40 min. However, neither intervention was protective in shorter, 20-min, periods of ischemia. Cardioprotection by both interventions was associated with increased activation of Akt, indicating the involvement of the RISK pathway. The early opening of an acutely occluded coronary artery is the treatment of choice in an evolving myocardial infarction [21]. However, initial successful opening, either spontaneously or after medical interventions, is several times followed by re-occlusion of the artery and this may negate any benefit obtained by the initial arterial opening. An additional protective intervention with short-lived periods of repetitive ischemic insults upon reperfusion may act as a postconditioning analog.
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Fig. 1. Schematic presentation of the study protocol.
The endogenous protective mechanisms of preconditioning and postconditioning reduce the final infarct size by decreasing the reperfusion injury [5]. Several experimental or clinical studies have extensively investigated the natural history [1,2,6] and the intracellular signaling [22,23] of these protective interventions but, to our knowledge, none of these studies tested their efficacy in two sequential periods of index ischemia. Preconditioning and postconditioning reduced the infarct size resulting from a 40-min ischemic insult, a finding that has already been reported by previous experimental studies [24–27]. We first explored the possibility of whether cardiac conditioning is feasible when two separate periods of 20-min ischemia with an intervening reperfusion interval occurred. We applied various modes of conditioning before, in-between and/or after the ischemic insults. All
interventions proved effective in limiting the infarct size and therefore we showed that cardiac conditioning is also feasible in this model of two sequential ischemic insults. In the present study, we confirmed the knowledge that shorter periods of ischemia cause substantially reduced infarct size compared to longer ones [28]. The effect of pre- and postconditioning did not reach statistical significance, in the 20 min ischemia subgroup because of the short duration of index ischemia. Both preconditioning and postconditioning need a sufficient burden of ischemia or a large area at risk in order to confer a considerable protection. Similar findings have also been reported from other studies in rats [29] and pigs [30], in which postconditioning failed to reduce the infarct size in the absence of an adequate burden of ischemia or a large area at risk [28]. Furthermore, we found that regional ischemia of a total 40-min duration resulted in similar infarct size,
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Table 1 Heart weight, blood pressure and heart rate of the different groups. Study group
HW
Baseline
20-min ischemia
180-min reperfusion
HR
BP
HR
BP
HR
BP
Infarct size analysis (n = 81) C-20 Prec-20 20-PostC C-40 Prec-40 40-PostC C-20–20 Prec-20–20 20–20-PostC 20-PostC-20 20-PostC-20-PostC Prec-20–20-PostC
7.7 ± 0.3 8.1 ± 0.5 7.3 ± 0.6 8.1 ± 0.4 8.2 ± 0.3 7.4 ± 0.5 7.2 ± 0.3 7.3 ± 0.4 7.0 ± 0.5 8.2 ± 0.6 7.2 ± 0.3 7.7 ± 0.2
272 ± 10 280 ± 11 285 ± 15 283 ± 12 276 ± 14 274 ± 13 282 ± 16 277 ± 13 278 ± 11 288 ± 10 270 ± 13 271 ± 10
82 ± 7 72 ± 4 80 ± 7 76 ± 5 80 ± 3 69 ± 7 74 ± 6 69 ± 6 77 ± 6 71 ± 3 79 ± 4 68 ± 3
278 ± 11 281 ± 15 280 ± 13 290 ± 11 273 ± 17 283 ± 13 280 ± 10 283 ± 14 268 ± 16 288 ± 13 270 ± 15 276 ± 14
74 ± 4 70 ± 2 77 ± 9 71 ± 7 79 ± 5 67 ± 6 68 ± 5 65 ± 4 71 ± 6 73 ± 7 71 ± 2 65 ± 2
254 ± 16 260 ± 13 255 ± 15 272 ± 16 266 ± 11 264 ± 17 269 ± 12 259 ± 15 261 ± 10 277 ± 14 262 ± 14 260 ± 14
68 ± 5 67 ± 4 67 ± 6 65 ± 7 72 ± 7 65 ± 8 65 ± 4 68 ± 6 72 ± 7 65 ± 6 66 ± 6 68 ± 5
Western blot analysis (n = 48) C-20 Prec-20 20-PostC C-40 Prec-40 40-PostC C-20–20 Prec-20–20 20–20-PostC 20-PostC-20 20-PostC-20-PostC Prec-20–20-PostC
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
283 ± 14 270 ± 12 275 ± 11 273 ± 14 279 ± 16 272 ± 11 272 ± 10 287 ± 14 273 ± 10 281 ± 15 279 ± 16 276 ± 14
81 ± 7 77 ± 3 68 ± 4 83 ± 7 73 ± 6 77 ± 8 69 ± 3 81 ± 7 77 ± 4 71 ± 4 69 ± 3 80 ± 7
283 ± 14 274 ± 13 279 ± 15 269 ± 10 281 ± 11 282 ± 14 269 ± 11 288 ± 11 283 ± 14 280 ± 12 274 ± 13 286 ± 11
72 ± 4 78 ± 4 69 ± 5 80 ± 6 73 ± 5 70 ± 4 64 ± 5 75 ± 9 69 ± 5 72 ± 5 66 ± 7 73 ± 7
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
HW: heart weight in g; HR: mean heart rate in beats/min; BP: mean blood pressure in mm Hg. 20-min ischemia is the period of the initial ischemic insult; and N/A: not applicable (no heart weight and not 180-min reperfusion in these groups).
independently of whether it was applied as a single 40-min period or two sequential 20-min periods. Thus, the total ischemic burden seems to be responsible for the final infarct size regardless of the way of its application. In other words, conditioning may not be effective in limiting the infarct size when the myocardium is not exposed to a sustained or a heavy burden of ischemia despite the activation of Akt. Apart from the effect of cardiac conditioning on infarct size, we further tried to shed some light on the intracellular changes that occurred in myocardium. It is known that among several mechanisms, conditioning confers protection via phosphorylation of several intracellular molecules that activate a series of specific kinases that in turn inhibit GSK3beta, hence preventing mitochondrial permeability transition pore (mPTP) opening and therefore cellular destruction. Among the most widely studied kinases are PI3 and Akt, which belong to the so-called RISKs [31]. Although several aspects of signal transduction in preconditioning and postconditioning are still under
Fig. 2. Percent infarct size to area at risk ratio (%I/R) in the different study groups (n = 81; * p b 0.05 versus C-40, # p b 0.05 versus C-20–20).
investigation, there is already a lot of evidence showing that both interventions share common, though identical, signaling pathways. It has been shown that adenosine, bradykinin, opioids and other ligands induce free radical release via PKC, RISKs, PKG and KATP channels [32]. Free radicals activate further PKC that in turn activates A2β receptors and another series of RISKs, finally preventing mPTP opening [32]. To complicate things, in addition to the above-mentioned mechanisms, alternative signaling molecules such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, connexin-43 or STAT3 are also involved in the mechanism of cardioprotection, despite the conflicting evidence in the literature [33]. It appears that there is a cross-talking between the different pathways and the increase of one mediator, which is critical for protection, may not be essential when another mediator promoting an alternative intracellular pathway is activated. In the present study, we found that Akt was activated in all groups with significant infarct size reduction, regardless of the protective intervention – preconditioning or postconditioning – or the timing of its application in the context of our study protocol. We also found that Akt was not activated either at baseline or in any group in which protection was not accomplished. Despite that several studies have shown that RISKs hold a key role in cardioprotection, there are other reports arguing against their significance or causal role at least in a pig model [23]. Taking under consideration that there is an extensive cross-talking between various triggers, mediators and end-effectors already discussed, the activation of a single kinase, Akt in the case of our study, does not necessarily implies a causative role, but it may only represent a indicator of cardioprotection. This is further supported by the fact that conditioning interventions activated Akt even after exposure to 20-min ischemia. As a result, Akt, along with several other kinases that are activated during cardiac conditioning, when studied independently, may only be considered as indicators of the intracellular changes taking place during protection. We assume that there may not be a single intracellular pathway responsible for the conferred protection. Despite the lack of statistically significant differences, preconditioning appeared consistently more protective than postconditioning in all
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Fig. 3. Western blot analysis of myocardial phospho-Akt and total Akt in the different study groups (*p b 0.05 versus baseline, C-20, C-40 and C-20–20).
experimental groups. This is in accordance with the reports having showed that postconditioning was less protective than preconditioning [10,28]. Preconditioning protects against both ischemic and reperfusion injury whereas postconditioning is only protective against the latter and thus preconditioning may be more efficient. We found that the amount of phosphorylated Akt is similar in both interventions. Combined preconditioning and postconditioning treatment protocols did not produce an additive protective effects in rats [13] or dogs [34], implying the presence of potentially overlapping mechanisms. However, combining preconditioning and postconditioning has been shown to induce an additive protection in rabbit and hence their mechanisms of action may be different [35]. It has been shown in rabbit hearts that ERK is not part of preconditioning's signaling [35,36]. Nevertheless the signal transduction of ischemic pre- and postconditioning is not yet entirely elucidated and different algorithms in postconditioning protocols may have yielded a different final infarct size [37]; this was however beyond the scope of the present study. In conclusion, according to our findings, conditioning of the heart by different models of intervention is feasible when two sequential periods of sustained ischemia occur. This piece of evidence may be clinically relevant in circumstances with two sequential ischemic insults. The associated activation of Akt may be a reliable marker of effective conditioning and not necessarily a crucial component of cardioprotection. Acknowledgments We thank ELPEN Pharmaceutical Co. Inc. for continued support. The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology. References [1] Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124–36. [2] Zhao Z-Q, Corvera JS, Halkos ME, et al. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol 2003;285:H579–88. [3] Przyklenk K, Bauer B, Ovize M, Kloner RA, Whittaker P. Regional ischemic ‘preconditioning’ protects remote virgin myocardium from subsequent sustained coronary occlusion. Circulation 1993;87:893–9.
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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Various models of cardiac conditioning in single or sequential periods of ischemia: Comparative effects on infarct size and intracellular signaling Konstantinos Iliodromitis a, Dimitrios Farmakis a, b, Ioanna Andreadou c,⁎, Anastasia Zoga a, Sofia-Iris Bibli c, Theodora Manolaki a, Nikolaos Dagres a, Efstathios K. Iliodromitis a, Maria Anastasiou-Nana a, Dimitrios Th. Kremastinos a a b c
Second Department of Cardiology, University of Athens Medical School, Attikon University Hospital, Athens, Greece First Department of Internal Medicine, University of Athens Medical School, Laiko Hospital, Athens, Greece Department of Pharmaceutical Chemistry, University of Athens School of Pharmacy, Athens, Greece
a r t i c l e
i n f o
Article history: Received 14 March 2012 Received in revised form 3 September 2012 Accepted 5 December 2012 Available online 25 December 2012 Keywords: Ischemia Reperfusion Infarct size Preconditioning Postconditioning Intracellular signaling
a b s t r a c t Background: Preconditioning (PreC) and postconditioning (PostC) reduce infarct size. We sought to determine the effects of PreC and PostC, alone or in combination, on infarct size and expression of intracellular signals in different ischemia models. Methods: Male rabbits were subjected to myocardial ischemia followed by 3-hour reperfusion. In a first series we applied 3 ischemia models [a 20-min period (20), a 40-min period (40), and two sequential 20-min periods (20–20)] and 3 types of interventions [no intervention (controls, C), 2 cycles of 5-min ischemia/10-min reperfusion before index ischemia (PreC) and 6 cycles of 10-s ischemia/10-s reperfusion after index ischemia and/or between the sequential ischemic periods (PostC)] (12 groups in total). Infarct size (I) and area at risk (R) were assessed (%I/R). In a second series, samples were taken for western blot analysis of Akt phosphorylation. Results: Overall, %I/R differed significantly among groups (p b 0.001). In control groups, C-40 had a greater %I/R than C-20 (p = 0.006). In intervention groups, no differences were found in %I/R. All intervention groups had significantly lower %I/R compared to C-40 group (p b 0.001), whereas, compared to C-20–20 group, PreC-20–20, 20-PostC-20, 20-PostC-20-PostC and PreC-20–20-PostC groups had lower %I/R (all p b 0.05). Akt was increased in all groups in which a significant %I/R reduction was achieved (p b 0.05 versus all other groups). Conclusions: PreC and PostC, alone or in combination, are effective when an ischemic insult of a given duration is applied either as a single or as sequential periods. Protection from either intervention is associated with an enhanced Akt activation. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Apart from the mechanical or pharmacological means of reperfusion that substantially limit myocardial necrosis during an evolving myocardial infarction, additional reduction of the final infarct size is feasible by conditioning the heart. Both preconditioning and postconditioning, local [1,2] or remote [3,4], have been extensively investigated and proved effective in preventing the reperfusion injury in all species studied so far, independently of the presence of collaterals in coronary circulation [5]. The initial experimental studies focused on the natural history of preconditioning and postconditioning [1,2,6], while later studies ⁎ Corresponding author at: Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens, Panepistimiopolis, Zografou, Athens 15771, Greece. Tel.: +30 210 7274827; fax: +30 210 7274747. E-mail address: [email protected] (I. Andreadou). 0167-5273/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijcard.2012.12.014
investigated the ligands and the intracellular transduction pathways in an attempt to substitute the mechanical means of conditioning with pharmacological agents [7–10]. The activation of the PI3K–Akt kinase pathway component of the Reperfusion Injury Salvage Kinase (RISK) pathway [11] at the onset of myocardial reperfusion has been implicated as a mediator of direct myocardial preconditioning [12] and postconditioning [13]. The acquired experience from the experimental studies has been successfully transferred into the clinical practice and various external interventions or pharmacological agents can successfully mimic conditioning and provide cardioprotection [14–18]. Following an initial ischemic insult resulting from coronary artery occlusion, a number of patients may suffer a second episode of sustained ischemia, either after a spontaneous or a therapeutic restoration of the flow. The role of pre- and postconditioning is unknown in these circumstances. Very few studies appear in the literature with two sequential periods of ischemia and to the best of our knowledge
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2.5. Western blot analysis
there is no study to test the effectiveness of these interventions when applied before, in-between and after the episodes of sustained ischemia with a parallel investigation of the intracellular mediators. In the present study, keeping in mind that several patients have a second episode of total coronary artery occlusion after an initially successful opening, we used an in vivo model of two separate and sequential periods of ischemia and reperfusion and we tested the effects of preconditioning and postconditioning on the infarct size and the RISK pathway after a single or two sequential episodes of ischemia and reperfusion.
In the second series of experiments, 48 animals were subjected to the above interventions (n = 4/group) up to the 5th min of reperfusion, when the hearts were quickly excised, immediately immersed into liquid nitrogen and then kept in −70 °C up to the time of Akt assessment. The phosphorylation state (phospho-Akt, Ser 473) and total levels of Akt were analyzed by SDS-PAGE immunoelectrophoresis using antibodies (Cell Signaling Technology) as previously described [20]. Relative densitometry was determined using a computerized software package (NIH Image) and the values for phospho-Akt were normalized to the values for total Akt.
2. Materials and methods
2.6. Statistical analysis
2.1. Animals
All results are presented as mean ± standard error of the mean. Variables were compared among the 12 groups using One-way Analysis of Variance (ANOVA) with Bonferroni correction and with Tukey post-hoc analysis. A p value b0.05 was considered as statistically significant.
A total of 143 New Zealand white male rabbits with a weight between 2.6 and 3.5 kg received proper care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institute of Health. Fourteen rabbits from different groups were excluded for various hemodynamic or technical reasons (severe pump failure during ischemia, intractable ventricular tachycardia/fibrillation, severe bleeding and infarction detected outside the area at risk). As a result, 129 animals completed the study. 2.2. Surgical procedures Sodium thiopentone at a dose of 30 mg/kg was injected into an ear vein for anesthesia. The animals were then intubated and connected to a respirator for small animals (MD Industries, Mobile, AL, USA) for mechanical ventilation at a rate adjusted to maintain normal blood gases. The experimental conditions have been previously described in detail [9,19]. A bipolar chest lead was used for continuous electrocardiographic monitoring. The chest was surgically opened with a left thoracotomy and the beating heart was exposed. The pericardium was opened and a 3-0 silk suture was passed around a prominent coronary artery. Ischemia was induced by pulling the thread through a small piece of soft tubing, which was firmly positioned against the coronary arterial wall with the aid of a small clamp. Ischemia resulted in ST elevation on the ECG and a change in the color of the myocardium. At the end of ischemic period, the snare was opened, the artery refilled and the myocardium reperfused [9,19]. 2.3. Experimental protocol The rabbits were randomly divided into 12 groups and subjected to regional myocardial ischemia followed by reperfusion. There were 3 models of ischemia [a 20-min period (20), a 40-min period (40), and two sequential 20-min periods separated by 12-min reperfusion (20–20)] and 3 types of intervention [(no intervention, controls, C), preconditioning with 2 cycles of 5-min ischemia/10-min reperfusion before index ischemia (PreC) and postconditioning with 6 cycles of 10-s ischemia/ 10-s reperfusion immediately after the index ischemia (PostC)]. The 12-min duration of the reperfusion interval was selected in order to allow comparison with the groups of preconditioning. In preconditioning, 10 min of reperfusion separates the last protective intervention of short ischemia/reperfusion from the index ischemia. Postconditioning with 6 cycles of 10-s ischemia/reperfusion account for an elapsed time of 2 min after the end of the first index ischemia. In order for all the groups to be comparable, 10 min of reperfusion was also required after the end of the last protective intervention of postconditioning and therefore a total time of 12 min was selected for the interval between the first and the second episode of sustained ischemia. The protocol is presented schematically in Fig. 1. 2.4. Risk zone and infarcted zone measurement In the first series of experiments, 81 rabbits (n = 6–8/group) followed the above protocol up to 3 h of reperfusion. At the end of reperfusion, hearts were excised, mounted on an apparatus and perfused with normal saline for 2 min for blood removal. Then the coronary ligature was retightened at the same site and 5 ml of green fluorescent microspheres were infused for the separation of the normally perfused area from the area at risk. Hearts were kept in the refrigerator for 24 h and cut into 3 mm thick sections. The slices were stained with triphenyltetrazolium chloride (TTC) at 37 °C and immersed in formaldehyde. With a wavelength of 366 nm UV light, we separated the risk from the infarcted zone of the heart and traced all the areas onto an acetate sheet in order to estimate the volumes of these areas in cm3. The tracings were subsequently scanned with the Adobe Photoshop 6.0 and measured with the Scion Image program. The areas of myocardial tissue at risk and infracted were automatically transformed into volumes. Infarct and risk area volumes were expressed in cm3 and the percent of infarct to risk area ratio (I/R) was calculated [9]. The evaluation of infarct size was performed by a researcher blinded to treatment assignment.
3. Results 3.1. Baseline characteristics Heart weight, blood pressure and heart rate of the different study groups are presented in Table 1. No significant differences were observed among the groups. 3.2. Infarct size Overall, I/R differed significantly among groups (pb 0.001). Post-hoc analyses in controls showed that C-40 group had greater I/R compared to C-20 group (43.2 ± 4.1% vs 24.5± 2.7%, p = 0.006), but not compared to C-20–20 group (34.6± 3.9%). Furthermore, I/R did not differ among the different intervention groups (13.7 ±1.2% in PreC-20, 20.6± 2.2% in 20-PostC, 19.9 ± 2.2% in PreC-40, 26.9 ± 2.4% in 40-PostC, 12.7 ± 3.5% in PreC-20–20, 20.1 ± 4.3% in 20–20-PostC, 15.7± 1.9% in 20-PostC-20 and 19.4 ±1.7% in Prec-20–20-PostC). When intervention and control groups were compared, all intervention groups had significantly lower I/R compared to C-40 group (pb 0.001), no intervention groups differed significantly compared to C-20 group, whereas compared to C-20–20 group, PreC-20–20, 20-PostC-20, 20-PostC-20-PostC and PreC-20–20-PostC groups had significantly lower I/R (p= 0.001, b0.001, 0.003, 0.011 and 0.036, respectively). The results are presented schematically in Fig. 2. 3.3. Western blot analysis The phosphorylation levels of Akt were significantly increased in all groups in which a significant I/R reduction was accomplished (Fig. 3, p b 0.05 versus all other groups). 4. Discussion The present study showed that both preconditioning and postconditioning, either alone or in combination, were effective in limiting the infarct size caused by either single or sequential periods of ischemia with a total duration of 40 min. However, neither intervention was protective in shorter, 20-min, periods of ischemia. Cardioprotection by both interventions was associated with increased activation of Akt, indicating the involvement of the RISK pathway. The early opening of an acutely occluded coronary artery is the treatment of choice in an evolving myocardial infarction [21]. However, initial successful opening, either spontaneously or after medical interventions, is several times followed by re-occlusion of the artery and this may negate any benefit obtained by the initial arterial opening. An additional protective intervention with short-lived periods of repetitive ischemic insults upon reperfusion may act as a postconditioning analog.
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Fig. 1. Schematic presentation of the study protocol.
The endogenous protective mechanisms of preconditioning and postconditioning reduce the final infarct size by decreasing the reperfusion injury [5]. Several experimental or clinical studies have extensively investigated the natural history [1,2,6] and the intracellular signaling [22,23] of these protective interventions but, to our knowledge, none of these studies tested their efficacy in two sequential periods of index ischemia. Preconditioning and postconditioning reduced the infarct size resulting from a 40-min ischemic insult, a finding that has already been reported by previous experimental studies [24–27]. We first explored the possibility of whether cardiac conditioning is feasible when two separate periods of 20-min ischemia with an intervening reperfusion interval occurred. We applied various modes of conditioning before, in-between and/or after the ischemic insults. All
interventions proved effective in limiting the infarct size and therefore we showed that cardiac conditioning is also feasible in this model of two sequential ischemic insults. In the present study, we confirmed the knowledge that shorter periods of ischemia cause substantially reduced infarct size compared to longer ones [28]. The effect of pre- and postconditioning did not reach statistical significance, in the 20 min ischemia subgroup because of the short duration of index ischemia. Both preconditioning and postconditioning need a sufficient burden of ischemia or a large area at risk in order to confer a considerable protection. Similar findings have also been reported from other studies in rats [29] and pigs [30], in which postconditioning failed to reduce the infarct size in the absence of an adequate burden of ischemia or a large area at risk [28]. Furthermore, we found that regional ischemia of a total 40-min duration resulted in similar infarct size,
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Table 1 Heart weight, blood pressure and heart rate of the different groups. Study group
HW
Baseline
20-min ischemia
180-min reperfusion
HR
BP
HR
BP
HR
BP
Infarct size analysis (n = 81) C-20 Prec-20 20-PostC C-40 Prec-40 40-PostC C-20–20 Prec-20–20 20–20-PostC 20-PostC-20 20-PostC-20-PostC Prec-20–20-PostC
7.7 ± 0.3 8.1 ± 0.5 7.3 ± 0.6 8.1 ± 0.4 8.2 ± 0.3 7.4 ± 0.5 7.2 ± 0.3 7.3 ± 0.4 7.0 ± 0.5 8.2 ± 0.6 7.2 ± 0.3 7.7 ± 0.2
272 ± 10 280 ± 11 285 ± 15 283 ± 12 276 ± 14 274 ± 13 282 ± 16 277 ± 13 278 ± 11 288 ± 10 270 ± 13 271 ± 10
82 ± 7 72 ± 4 80 ± 7 76 ± 5 80 ± 3 69 ± 7 74 ± 6 69 ± 6 77 ± 6 71 ± 3 79 ± 4 68 ± 3
278 ± 11 281 ± 15 280 ± 13 290 ± 11 273 ± 17 283 ± 13 280 ± 10 283 ± 14 268 ± 16 288 ± 13 270 ± 15 276 ± 14
74 ± 4 70 ± 2 77 ± 9 71 ± 7 79 ± 5 67 ± 6 68 ± 5 65 ± 4 71 ± 6 73 ± 7 71 ± 2 65 ± 2
254 ± 16 260 ± 13 255 ± 15 272 ± 16 266 ± 11 264 ± 17 269 ± 12 259 ± 15 261 ± 10 277 ± 14 262 ± 14 260 ± 14
68 ± 5 67 ± 4 67 ± 6 65 ± 7 72 ± 7 65 ± 8 65 ± 4 68 ± 6 72 ± 7 65 ± 6 66 ± 6 68 ± 5
Western blot analysis (n = 48) C-20 Prec-20 20-PostC C-40 Prec-40 40-PostC C-20–20 Prec-20–20 20–20-PostC 20-PostC-20 20-PostC-20-PostC Prec-20–20-PostC
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
283 ± 14 270 ± 12 275 ± 11 273 ± 14 279 ± 16 272 ± 11 272 ± 10 287 ± 14 273 ± 10 281 ± 15 279 ± 16 276 ± 14
81 ± 7 77 ± 3 68 ± 4 83 ± 7 73 ± 6 77 ± 8 69 ± 3 81 ± 7 77 ± 4 71 ± 4 69 ± 3 80 ± 7
283 ± 14 274 ± 13 279 ± 15 269 ± 10 281 ± 11 282 ± 14 269 ± 11 288 ± 11 283 ± 14 280 ± 12 274 ± 13 286 ± 11
72 ± 4 78 ± 4 69 ± 5 80 ± 6 73 ± 5 70 ± 4 64 ± 5 75 ± 9 69 ± 5 72 ± 5 66 ± 7 73 ± 7
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
HW: heart weight in g; HR: mean heart rate in beats/min; BP: mean blood pressure in mm Hg. 20-min ischemia is the period of the initial ischemic insult; and N/A: not applicable (no heart weight and not 180-min reperfusion in these groups).
independently of whether it was applied as a single 40-min period or two sequential 20-min periods. Thus, the total ischemic burden seems to be responsible for the final infarct size regardless of the way of its application. In other words, conditioning may not be effective in limiting the infarct size when the myocardium is not exposed to a sustained or a heavy burden of ischemia despite the activation of Akt. Apart from the effect of cardiac conditioning on infarct size, we further tried to shed some light on the intracellular changes that occurred in myocardium. It is known that among several mechanisms, conditioning confers protection via phosphorylation of several intracellular molecules that activate a series of specific kinases that in turn inhibit GSK3beta, hence preventing mitochondrial permeability transition pore (mPTP) opening and therefore cellular destruction. Among the most widely studied kinases are PI3 and Akt, which belong to the so-called RISKs [31]. Although several aspects of signal transduction in preconditioning and postconditioning are still under
Fig. 2. Percent infarct size to area at risk ratio (%I/R) in the different study groups (n = 81; * p b 0.05 versus C-40, # p b 0.05 versus C-20–20).
investigation, there is already a lot of evidence showing that both interventions share common, though identical, signaling pathways. It has been shown that adenosine, bradykinin, opioids and other ligands induce free radical release via PKC, RISKs, PKG and KATP channels [32]. Free radicals activate further PKC that in turn activates A2β receptors and another series of RISKs, finally preventing mPTP opening [32]. To complicate things, in addition to the above-mentioned mechanisms, alternative signaling molecules such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, connexin-43 or STAT3 are also involved in the mechanism of cardioprotection, despite the conflicting evidence in the literature [33]. It appears that there is a cross-talking between the different pathways and the increase of one mediator, which is critical for protection, may not be essential when another mediator promoting an alternative intracellular pathway is activated. In the present study, we found that Akt was activated in all groups with significant infarct size reduction, regardless of the protective intervention – preconditioning or postconditioning – or the timing of its application in the context of our study protocol. We also found that Akt was not activated either at baseline or in any group in which protection was not accomplished. Despite that several studies have shown that RISKs hold a key role in cardioprotection, there are other reports arguing against their significance or causal role at least in a pig model [23]. Taking under consideration that there is an extensive cross-talking between various triggers, mediators and end-effectors already discussed, the activation of a single kinase, Akt in the case of our study, does not necessarily implies a causative role, but it may only represent a indicator of cardioprotection. This is further supported by the fact that conditioning interventions activated Akt even after exposure to 20-min ischemia. As a result, Akt, along with several other kinases that are activated during cardiac conditioning, when studied independently, may only be considered as indicators of the intracellular changes taking place during protection. We assume that there may not be a single intracellular pathway responsible for the conferred protection. Despite the lack of statistically significant differences, preconditioning appeared consistently more protective than postconditioning in all
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Fig. 3. Western blot analysis of myocardial phospho-Akt and total Akt in the different study groups (*p b 0.05 versus baseline, C-20, C-40 and C-20–20).
experimental groups. This is in accordance with the reports having showed that postconditioning was less protective than preconditioning [10,28]. Preconditioning protects against both ischemic and reperfusion injury whereas postconditioning is only protective against the latter and thus preconditioning may be more efficient. We found that the amount of phosphorylated Akt is similar in both interventions. Combined preconditioning and postconditioning treatment protocols did not produce an additive protective effects in rats [13] or dogs [34], implying the presence of potentially overlapping mechanisms. However, combining preconditioning and postconditioning has been shown to induce an additive protection in rabbit and hence their mechanisms of action may be different [35]. It has been shown in rabbit hearts that ERK is not part of preconditioning's signaling [35,36]. Nevertheless the signal transduction of ischemic pre- and postconditioning is not yet entirely elucidated and different algorithms in postconditioning protocols may have yielded a different final infarct size [37]; this was however beyond the scope of the present study. In conclusion, according to our findings, conditioning of the heart by different models of intervention is feasible when two sequential periods of sustained ischemia occur. This piece of evidence may be clinically relevant in circumstances with two sequential ischemic insults. The associated activation of Akt may be a reliable marker of effective conditioning and not necessarily a crucial component of cardioprotection. Acknowledgments We thank ELPEN Pharmaceutical Co. Inc. for continued support. The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology. References [1] Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124–36. [2] Zhao Z-Q, Corvera JS, Halkos ME, et al. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol 2003;285:H579–88. [3] Przyklenk K, Bauer B, Ovize M, Kloner RA, Whittaker P. Regional ischemic ‘preconditioning’ protects remote virgin myocardium from subsequent sustained coronary occlusion. Circulation 1993;87:893–9.
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