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Eur J Cardiothorac Surg 2005;28:821-831
© 2005 Elsevier Science NL
Cardiac Surgical Research/Cardiothoracic Surgery, The Rayne Institute, Guy's and St Thomas’ NHS Foundation Trust, St Thomas’ Hospital, London SE1 7EH, UK
Received 4 April 2005; received in revised form 14 June 2005; accepted 27 June 2005.
* Corresponding author. Tel.: +44 20 7261 0157; fax: +44 20 7928 0658. (Email: david.chambers{at}kcl.ac.uk).
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Abstract |
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 Top Abstract 1. Introduction2. Material and methods3. Results4. Discussion5. ConclusionReferences |
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Key Words: Research • Ischemia • Preconditioning • Left ventricular function • Heart preservation
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1. Introduction |
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TopAbstract1. Introduction2. Material and methods3. Results4. Discussion5. ConclusionReferences |
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It has been known for some time [5] that single or multiple brief periods of myocardial ischemia or hypoxia trigger an adaptive response that protects the heart against injury from a subsequent prolonged period of ischemia and reperfusion. This phenomenon, known as ischemic preconditioning (IPC), has been demonstrated [5] to be effective against myocardial ischemic injury (infarct size or deterioration in function) and reperfusion-induced arrhythmias in a variety of different models and species (including man). Various intracellular signaling pathways have been implicated in the protective mechanism of IPC, and a number of different mediators have been shown to be involved in the IPC process, including adenosine receptors, protein kinase C (PKC), and ATP-sensitive potassium channel (KATP-channel) [5].
As a result of studies investigating the phenomenon of ischemic preconditioning, there has been renewed interest in ICCF as a cardioprotective procedure, with speculation that the protection involves an ischemic preconditioning mechanism. Indeed, Abd-Elfattah and colleagues [6] demonstrated significant maintenance of myocardial ATP levels in hearts subjected to short intermittent global ischemia periods compared to prolonged ischemia in dog hearts, and this has been confirmed in clinical studies [7]. We speculated in our previous ICCF study [4] that ischemic preconditioning may be involved in ICCF protection; however, that study was not designed to specifically address that question. In this study, the involvement of a preconditioning mechanism in ICCF protection is examined using drugs to target putative components of the preconditioning pathway, the KATP-channel and PKC.
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2. Material and methods |
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TopAbstract1. Introduction2. Material and methods3. Results4. Discussion5. ConclusionReferences |
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2.2 Heart isolation, perfusion and perfusion medium
Hearts were immediately excised from the anesthetized rat and immersed in cold (4 °C) Krebs–Henseleit buffer. The aorta was then rapidly cannulated, and the heart perfused in the Langendorff mode (at a constant pressure of 75 mmHg and at 37 °C) as previously described [4]. All hearts were subjected to an equilibration period of aerobic perfusion for 20 min, and baseline readings of left ventricular systolic pressure (LVSP: in mmHg), left ventricular end-diastolic pressure (LVEDP: in mmHg), heart rate (beats/min) and coronary flow rate (mL/min) were then taken. Left ventricular developed pressure (LVDP) was calculated as the LVSP minus LVEDP. Coronary flow rate was measured by timed collection into a measuring cylinder of the coronary effluent exiting the heart.
The perfusion medium was a modified Krebs Henseleit bicarbonate buffer (KHB) with the following composition (mmol/L): NaCl, 118.5; NaHCO3, 25.0; KCl, 4.8; MgSO4, 1.2; KH2PO4, 1.2; CaCl2, 1.4; and glucose, 11.0. The buffer was prepared daily, filtered through a 5-µm pore-size cellulose nitrate membrane filter before use, and continuously gassed with a mixture of 95% O2:5% CO2 to give a pH of 7.4 at 37 °C.
2.3 Exclusion criteria
Hearts not satisfying pre-assigned exclusion criteria at the time of the baseline readings (after 20 min of aerobic perfusion) were excluded from the study. The acceptable ranges for LVDP, heart rate, and coronary flow rate were >100 mmHg, >220 beats/min, and 8–16 mL/min, respectively.
2.4 Induction and termination of ventricular fibrillation
VF was induced by an electrical fibrillator (model G570, Department of Bioengineering, St Thomas’ Hospital) by passing alternating current through two electrodes. The silver electrodes were coated with silicone except for the final cm; one was attached to the apex of the ventricle and the other to the aortic cannula for grounding. The minimum voltage necessary to achieve an alternating current that maintained VF was used. If VF occurred during reperfusion or did not terminate spontaneously after the fibrillator was turned off, it was terminated by the use of a defibrillator (model G434, Department of Bioengineering, St Thomas’ Hospital).
2.5 Drugs
Glibenclamide (Glib: provided as a gift from Aventis Pharma, Tokyo, Japan) was dissolved in dimethyl sulfoxide (DMSO; 0.1% final concentration in all drug groups) and diluted in perfusion solution to obtain a final drug concentration of 10 µmol/L immediately before use. 5-Hydroxydecanoate (5HD; Sigma-Aldrich, Poole, Dorset, UK) was dissolved in deionized water to make a 50 mmol/L stock solution, the stock solution was diluted in perfusion solution to obtain a final drug concentration of 50 µmol/L immediately before use. Chelerythrine (Che; Sigma-Aldrich, Poole, Dorset, UK) was dissolved in deionized water to make a 2 mmol/L stock solution, the stock solution was diluted in perfusion solution to obtain a final drug concentration of 2 µmol/L immediately before use. The final concentrations of the drugs used in this study were taken from a consensus of the literature in which these drugs have been used.
2.6 Experimental protocols
All hearts were subjected to an equilibration period of aerobic perfusion at 37 °C for 20 min with KHB. Three studies were conducted (Fig. 1
).
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After equilibration, hearts were randomized to one of five groups (n = 6 hearts/group): Group 1: Control, 30 min additional aerobic perfusion with KHB followed by 30 min global 37 °C ischemia; Group 2: DMSO, 30 min additional aerobic perfusion with KHB containing 0.1% DMSO followed by 30 min global 37 °C ischemia; Group 3: Glib, 30 min additional aerobic perfusion with KHB containing Glib (10 µmol/L) followed by 30 min global 37 °C ischemia; Group 4: ICCFx1, 30 min additional aerobic perfusion with KHB, then a 10 min episode of global 37 °C ischemia with electrically-induced VF, followed by 10 min of reperfusion in sinus rhythm (SR) followed by 30 min global 37 °C ischemia; Group 5: ICCFx1 + Glib, 30 min additional aerobic perfusion with KHB containing Glib (10 µmol/L), then a 10 min episode of global 37 °C ischemia with electrically induced VF, followed by 10 min of reperfusion in SR with KHB containing Glib (10 µmol/L) followed by 30 min global 37 °C ischemia. LVEDP (intracavity pressure) was measured throughout these protocols. All groups were then subjected to 60 min of reperfusion at 37 °C with KHB, when recovery of myocardial function (LVDP, LVEDP, coronary flow rate) and myocardial injury (creatine kinase leakage) were measured.
2.6.2 Study 2
This study was designed to determine whether the cardioprotection induced by an ICCF protocol similar to that used clinically during myocardial revascularization operations (typically 4 x 10 min episodes of ICCF with 10 min reperfusion in sinus rhythm (SR) and no prolonged ischemic duration) involves a preconditioning mechanism associated with the opening of KATP-channels as mediator. This was examined by administering the non-specific KATP-channel blocker, glibenclamide, to bracket the ICCF episodes and determine whether it inhibits the potential mediator. As with our previous study [4], we were also interested in comparing the cumulative ICCF duration (40 min) to a continuous period of global ischemia of the same duration and whether glibenclamide influenced injury.
After equilibration, hearts were randomized to one of five groups (n = 6 hearts/group): Group 1: ICCFx4 [as Control], 30 min additional aerobic perfusion with KHB then four intermittent episodes of 10 min global 37 °C ischemia with electrically induced VF followed by 10 min reperfusion in SR; Group 2: ICCFx4 + Glib, 30 min additional aerobic perfusion with KHB containing Glib (10 µmol/L) then four intermittent episodes of 10 min global 37 °C ischemia with electrically induced VF followed by 10 min reperfusion in SR with KHB containing Glib (10 µmol/L); Groups 3–5: Ischemia, DMSO and Glib, respectively, as for Study 1 except that continuous global 37 °C ischemia for 40 min. LVEDP (intracavity pressure) was measured throughout these protocols. All groups were then subjected to 60 min of reperfusion at 37 °C with KHB, when recovery of myocardial function (LVDP, LVEDP, coronary flow rate) and myocardial injury (creatine kinase leakage) were measured.
2.6.3 Study 3
In this study, the potential preconditioning mechanism was examined in more detail by administering drugs shown to be associated with targeting specific putative components of the preconditioning signaling pathway(s) to bracket the ICCF episodes. We used chelerythrine to inhibit protein kinase C activity and 5-hydroxydecanoate (5HD) to block the mitochondrial KATP-channel. We also investigated the effects of these blockers on injury induced by continuous global ischemia of the same duration as the cumulative ICCF ischemia (40 min).
After equilibration, hearts were randomized to one of six groups (n = 6 hearts/group): Group 1: ICCFx4 [as Control], as for Study 2; Group 2: ICCFx4 + Che, 20 min additional aerobic perfusion with KHB then 10 min aerobic perfusion with KHB containing Che (2 µmol/L) followed by four intermittent episodes of 10 min global 37 °C ischemia with electrically induced VF and 10 min reperfusion in SR with KHB containing Che (2 µmol/L); Group 3: ICCFx4 + 5HD, 30 min additional aerobic perfusion with KHB containing 5HD (50 µmol/L) then four intermittent episodes of 10 min global 37 °C ischemia with electrically induced VF followed by 10 min reperfusion in SR with KHB containing 5HD (50 µmol/L); Group 4: Ischemia, as for Study 2; Group 5: Che, 20 min additional aerobic perfusion with KHB then 10 min aerobic perfusion with KHB containing Che (2 µmol/L) followed by continuous global 37 °C ischemia for 40 min; Group 6: 5HD, 30 min additional aerobic perfusion with KHB containing 5HD (50 µmol/L) followed by continuous global 37 °C ischemia for 40 min. LVEDP (intracavity pressure) was measured throughout these protocols. All groups were then subjected to 60 min of reperfusion at 37 °C with KHB, when recovery of myocardial function (LVDP, LVEDP, coronary flow rate), myocardial injury (creatine kinase leakage) and myocardial viability were measured.
2.7 Expression of results
Postischemic recovery of LVDP was expressed as a percentage of the baseline values (taken at the end of 20 min aerobic perfusion); LVEDP was expressed as absolute values (mmHg).
2.8 Creatine kinase (CK) leakage
CK leakage was measured as a marker of myocardial injury. In each protocol, total CK leakage (expressed as IU per g heart wet weight per min of total reperfusion duration) was assessed by spectrophotometric analysis of enzyme activity in the coronary effluent collected during all reperfusion periods, using a commercially available kit (Sigma-Aldrich Diagnostic Kits, Poole, Dorset, UK).
2.9 Triphenyltetrazolium chloride (TTC) assay
An assay of reduced TTC (formazan, dissolved in DMSO) was used as an index of myocardial viability (modified from an original MTT assay used for assessing viability of myocardial biopsies). In Protocol 3, after 60 min of reperfusion, hearts were immediately frozen in liquid nitrogen and stored for later analysis. The frozen heart was placed in a conical tube and minced into small pieces in 20 mL of phosphate buffer solution containing TTC (at a final concentration of 1 mmol/L) and incubated for 90 min at 37 °C. The buffer was decanted and the heart tissue was homogenized in 5 mL DMSO and agitated at a shaking table for 90 min. The homogenate was then centrifuged at 10,000 rpm for 1 min, the supernatant was withdrawn and the absorbance measured spectrophotometrically at 480 nm and the results expressed as arbitrary units/g heart wet weight.
2.10 Statistics
Statistical analysis was performed with StatView (SAS Institute Inc., NC, USA) on an Apple Macintosh computer. All data are reported as mean ± SEM. Comparisons between groups were assessed for significance by analysis of variance (ANOVA) or repeated measures ANOVA, as appropriate; if significance was established, post hoc analysis was assessed by the Student–Newman–Keuls test, which allowed for multiple comparisons. Within group comparisons were assessed by unpaired t-tests, as appropriate. A value of p
< 0.05 was considered statistically significant.
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3. Results |
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TopAbstract1. Introduction2. Material and methods3. Results4. Discussion5. ConclusionReferences |
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3.2.1.3 CK leakage (myocardial injury)
Total CK leakage (Fig. 3A) was similar in Control, DMSO and Glib hearts. In the ICCF group of hearts, CK leakage was significantly reduced, whereas Glib treatment in the ICCF group prevented this reduction. In the ICCF groups, CK leakage during the initial reperfusion period (Fig. 3B) was minimal, and most CK leakage occurred after the prolonged ischemic period; however, Glib treatment significantly increased CK leakage at both periods, mirroring the poorer recovery in these hearts.
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3.3.1.3 CK leakage (myocardial injury)
Total CK leakage (Fig. 5A) was significantly lower in the ICCF group compared to all other groups, which had similar levels of CK leakage. Fig. 5B shows CK release in both ICCF groups after each ICCF episode; the highest release occurred during the final prolonged reperfusion period. In the ICCF group, there were no differences in CK leakage during reperfusion after ICCF periods 1, 2 and 3, but it was significantly higher during the final prolonged reperfusion period. Glib treatment significantly elevated CK release at each period when compared to ICCF hearts, mirroring the reduced recovery of function in this group.
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3.4.1.3 CK leakage (myocardial injury)
Total CK leakage is shown in Fig. 7A; CK leakage in the ICCF hearts was significantly lower than all other groups. Although Che and 5HD treatment with ICCF induced nominally higher CK leakage, these values were not different from the control groups. Fig. 7B shows the CK leakage in the ICCF groups during each reperfusion period; CK leakage increased after each ICCF episode and was maximal during the final, prolonged, reperfusion period. As with Study 2, CK leakage during reperfusion after ICCF episodes 1, 2 and 3 were not different in the ICCF group, but there was a significant increase during the final reperfusion period. CK leakage was significantly higher in the treated ICCF groups after ICCF episodes 2–4, again mirroring the reduced recovery of function in these groups.
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reading/g wet wt. of 0.68 ± 0.03) and was significantly (p
< 0.01) higher than all other groups. However, hearts from the 5HD treatment with ICCF group also showed a significantly increased viability (0.47 ± 0.05; p
< 0.01) compared to the ischemia control group (0.26 ± 0.02). There were no significant differences between values in all other groups. |
4. Discussion |
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TopAbstract1. Introduction2. Material and methods3. Results4. Discussion5. ConclusionReferences |
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The efficacy of non-cardioplegic methods of cardiac protection during surgery, including intermittent aortic cross-clamping, with or without ventricular fibrillation, have been documented both experimentally [4] and clinically [9] in elective primary and reoperative CABG patients, as well as in higher risk patients [10]. Casthely and colleagues [11] confirmed these results by echocardiographic analysis of systolic and diastolic function and speculated that myocardial preservation using an ICCF technique may involve the phenomenon of ischemic preconditioning. In retrospect, a number of earlier studies have supported this contention. It was demonstrated [5] that four brief periods of regional myocardial ischemia prevented cumulative metabolic deficits and ischemic cell death in dog hearts. Similarly, in a study of cardiopulmonary bypass in dogs, Abd-Elfattah and colleagues [6] showed that myocardial ATP levels were maintained after a second episode of an intermittent global ischemia and reperfusion protocol and were significantly higher at the end than after the same cumulative, but sustained, ischemic duration. In the human heart undergoing CABG, myocardial ATP levels in control ICCF patients were reported [12] to be maintained at the same level as the preconditioned patients after the initial 10 min ICCF episode despite the absence of a pre-ICCF preconditioning stimulus. This preservation of myocardial high-energy phosphates is similar to that seen in early ischemic preconditioning studies [5] suggesting that the initial ischemia and reperfusion episode activated an endogenous protective mechanism (that may be preconditioning) and induced the protective effect against subsequent episodes. Interestingly, further studies in patients undergoing cardiac surgery [5] demonstrated that a specific preconditioning protocol (2 x 3 min aortic cross-clamping separated by 2 min reperfusion) prior to ICCF induced a lower release of troponin T than either ICCF or cardioplegic protection, although no other clinical effects were observed. Pharmacological preconditioning (using a selective adenosine A1 receptor agonist, GR79236X) before ICCF, however, was not effective [5].
Ischemic preconditioning is a phenomenon whereby one or more brief episodes of ischemia induce increased myocardial tolerance to subsequent prolonged ischemic insults, and this has been confirmed in many different animal models [5]. The precise mechanism of IPC remains controversial but opening of the KATP-channel has been proposed as one of the common effector mechanisms involved in IPC protection, although how the opening of these channels initiates cardioprotection is unknown. KATP-channel openers reduce ischemic damage [13] and the involvement of the KATP-channel in IPC has been demonstrated in many species [5,8] including dog, pig and rabbit using a non-specific KATP-channel blocker, glibenclamide. Controversially, early studies [14] were unable to demonstrate the involvement of the KATP-channel in IPC protection of infarct size or contractile function in the rat heart when glibenclamide was used as a KATP-channel blocker. This controversy was resolved, however, when it was realized that the inhibitory effect of glibenclamide to block IPC was time-dependent, with a slow onset of action, confirming the involvement of the KATP-channel in IPC in the rat heart. In this study, we were able to demonstrate that glibenclamide, when administered 30 min before the ICCF procedure in the isolated rat heart, attenuated the cardioprotective effect of ICCF, suggesting the involvement of the KATP-channel in the protective efficacy of ICCF.
More recently, IPC protection has been suggested to occur via activation of a mitochondrial KATP-channel (mito-KATP) [5,15], since shortening of the action potential by opening KATP-channels did not necessarily correlate with cardioprotection [15]. Subsequently, Garlid and co-workers [16] demonstrated, in rat hearts, that the mito-KATP-channel was a possible site of action for the cardioprotective effects of KATP-channel openers. In addition, they showed that the selective mito-KATP-channel antagonist, 5-hydroxydecanoic acid (5HD), completely abolished the cardioprotective effects of the potent mito-KATP-channel opener, diazoxide. Various studies have shown [17,18] that inhibition of the mito-KATP-channel by 5HD attenuates IPC. Fryer and colleagues [18] reported that in vivo intravenous administration of 5HD in Wistar rats at 10 or 30 min before IPC did not reduce cardioprotection whereas partial attenuation was seen when given 5 min before IPC; thus, 5HD metabolism may be rapid in the in vivo rat myocardium. Infarct size reduction by pharmacological (adenosine) preconditioning in Langendorff-perfused rabbit hearts was attenuated by 5HD administered only 2 min before and during the adenosine infusion and during washout [17], and Schultz and colleagues [19] demonstrated that infarct size reduction induced by IPC was completely abolished by 5HD in intact rats, when given intravenously 15 min prior to IPC. In contrast, Grover and coworkers [20] were unable to show that 5HD infused 10 min before and during IPC stimulus episodes abolished ischemic preconditioning in isolated rat heart, when recovery of function and LDH release were used as endpoints. In this study, 5HD was infused for 30 min before the start of ICCF and during the ICCF procedure and we demonstrated that 5HD attenuated the enhanced protection (recovery of function) and the reduction in CK release of ICCF; however, increased TTC staining was only partially reduced which may indicate rapid metabolism during the reperfusion period. Our results suggest that the mito-KATP-channel appears to be involved in the cardioprotective effect of ICCF.
Activation of PKC has been shown [5,21] to be central to the cardioprotection induced by ischemic preconditioning, either on infarct size reduction in rabbit or rat hearts or on improved recovery of function in rat hearts [5]. PKC activation by agents such as diacylglycerol (the potent physiological PKC activator) mimicked the protection induced by ischemia, whereas inhibitors of PKC activation, such as staurosporine, polymixin B or chelerythrine, abolished preconditioning protection. It is now accepted that PKC plays an important role in IPC. This study demonstrated that ICCF improved the recovery of function, attenuated myocardial injury and preserved myocardial viability and that this protective effect was reduced by chelerythrine, a specific antagonist of PKC. These findings suggest that PKC is involved in the effective protection induced by ICCF. However, it has been demonstrated [22] that PKC activity is not necessary during the trigger ischemic episode but is required during the subsequent prolonged ischemia. Inhibition of PKC activity with staurosporine for 5 min before and for 10 min of the prolonged ischemia inhibited the protection. In the ICCF procedure, there is no prolonged ischemia and it remains to be established whether the second ICCF episode (of 10 min duration) is sufficient to activate PKC. It is possible that inhibition of PKC prior to this second ICCF episode alone would have been sufficient to block ICCF (and hence IPC) protection but this was not examined in this study. Clinically, a preconditioning trigger prior to ICCF reduced troponin T release, suggesting that the 10 min episode of ICCF was a sufficiently prolonged ischemic duration to act as the index ischemia [5].
It has been shown [23] that either a single episode or repeat (two-cycle) episodes of IPC confer equal degrees of cardioprotection, although Sandhu and coworkers [24] reported that multiple (three-cycle) episodes of IPC provided more effective protection against myocardial necrosis than one IPC episode. These investigators also showed differences in protection and susceptibility to blockade by PKC inhibitors and suggested that repetitive IPC episodes may activate more pathways within the IPC cascade than a single IPC stimulus. In contrast, a recent study from our laboratory has demonstrated that increasing the number of ICCF episodes (thereby increasing the cumulative ischemic duration) significantly decreases the recovery of LVDP. Sunderdiek and coworkers [25], in a clinical study, documented that the technique of ICCF seemed less effective when the total ischemic duration was longer than 40 min in comparison to cardioplegia. The reasons for this discrepancy remain unclear, but the most obvious difference is the absence of the prolonged ischemic period after the preconditioning stimulus in ICCF procedures.
4.1 Limitations of the study
In the present study, our findings were made in the normal healthy rat heart. Therefore, we could not evaluate the effect of maldistribution of chelerythrine, glibenclamide and 5HD, or any potential disadvantage of ICCF in the diseased heart. It should be noted, however, that ICCF is successfully used clinically in diseased hearts and may have advantages over cardioplegia in avoiding effects of maldistribution. There is also the possibility that the effects described in these studies are species specific (to rat hearts) and that these findings may not be applicable to the human heart. However, preconditioning has been shown to be possible in human hearts [7,12] and myocardial ATP levels were maintained in patients subjected to ICCF [12].
Another possible limitation of the present study may be the use of an isolated heart preparation that does not possess a collateral circulation. The validity of the explanation for these results relies on the specificity of the PKC inhibitor, chelerythrine, and the mito-KATP-channel blocker 5HD. Although the concentration of these drugs used in this study is known to be effective for inhibition of PKC and mito-KATP-channel, respectively, the implication of non-specific effects of these drugs in modulating cardioprotection by ICCF cannot be ruled out.
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5. Conclusion |
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TopAbstract1. Introduction2. Material and methods3. Results4. Discussion5. ConclusionReferences |
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Acknowledgments |
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References |
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TopAbstract1. Introduction2. Material and methods3. Results4. Discussion5. ConclusionReferences |
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This article has been cited by other articles:
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  M. Scarci, H. B. Fallouh, C. P. Young, and D. J. Chambers Does intermittent cross-clamp fibrillation provide equivalent myocardial protection compared to cardioplegia in patients undergoing bypass graft revascularisation? Interactive CardioVascular and Thoracic Surgery, November 1, 2009; 9(5): 872 - 878. [Abstract] [Full Text] [PDF]
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 S. R. Walsh, T. Y. Tang, P. Kullar, D. P. Jenkins, D. P. Dutka, and M. E. Gaunt Ischaemic preconditioning during cardiac surgery: systematic review and meta-analysis of perioperative outcomes in randomised clinical trials Eur. J. Cardiothorac. Surg., November 1, 2008; 34(5): 985 - 994. [Abstract] [Full Text] [PDF]
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P. K. Mishra Fibrillatory arrest technique: is it worth tasting the old wine in new bottle? Eur. J. Cardiothorac. Surg., May 1, 2006; 29(5): 860 - 860. [Full Text] [PDF]
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J. Vaage Editorial comment: Old skills in a new context--But do we want to use it? Eur. J. Cardiothorac. Surg., December 1, 2005; 28(6): 831 - 832. [Full Text] [PDF]
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p
< 0.01 compared with other ICCF values.
p
< 0.01 compared with control groups.