Dm Yellon

ADENOSINE RECEPTOR INVOLVEMENT IN A DELAYED PHASE OF MYOCARDIAL PROTECTION 24 HOURS AFTER ISCHEMIC PRECONDITIONING

BackgroundWe previously reported a delayed phase of protection against infarction 24 hours after ischemic preconditioning in the rabbit. In the present study, we investigated the possibility that this “second window of protection,” like the well-described early phase of protection in the rabbit, might be associated with adenosine receptor activation. Methods and ResultsIn the first series of experiments, we examined whether adenosine receptor blockade with 8-(p-sulfophenyl)- theophylline (SPT) during preconditioning could abolish the delayed protection against infarction 24 hours later. Open-chest rabbits were subjected to myocardial preconditioning (PC) with the four 5-minute coronary occlusions or they were sham operated on (SHAM). During these procedures, animals received either SPT (PC + SPT, n = 6; and SHAM + SPT, n = 6) or vehicle (PC + VEH, n = 12; and SHAM + VEH, n = 11). Twenty-four hours later, infarct development after a 30-minute coronary occlusion/120-minute reperfusion insult was assessed with triphenyltetrazolium staining. In vehicle-treated rabbits, the infarct-to-risk ratio (I/R) was reduced from 53.6 ± 5.7% (SHAM + VEH) to 32.9 ± 4.6% (PC + VEH) (P < .05), clearly indicating a delayed phase of protection. Although I/R was not significantly different between SHAM + VEH (53.6 ± 5.7%) and SHAM + SPT (61.7 ± 5.4%), in PC + SPT the delayed protection was abolished (I/R = 56.8 ± 3.8%). In the second series of experiments, we examined if pharmacological adenosine A1 receptor stimulation could evoke a delayed phase of protection. Conscious rabbits were pretreated with intravenous boluses of saline or the A1 receptor–selective agonist 2-chloro-N6-cyclopentyladenosine (CCPA), and infarct size in response to 30-minute ischemia/120-minute reperfusion was assessed 24 hours later. I/R was 54.5 ± 2.7% in saline-pretreated controls (n = 12). Pretreatment with 25 μg/kg CCPA (n = 6), 50 μg/kg CCPA (n = 6), or 100 μg/kg CCPA (n = 6) resulted in I/R ratios of 37.1 ± 4.2% (P < .01), 37.7 ± 2.2% (P < .01), and 26.3 ± 5.7% (P < .01), respectively. In both series of experiments, there were no differences in systemic hemodynamics during the infarct protocol, assessed as rate-pressure product, between the different experimental groups. ConclusionsTwenty-four hours after repetitive brief coronary occlusions, susceptibility to infarction in rabbit myocardium is reduced, an effect that may have clinical relevance. Results of the present study suggest that this second window of protection following preconditioning may, like the early phase of protection, be initiated by an adenosine-related mechanism.

Ischaemic preconditioning reduces troponin T release in patients undergoing coronary artery bypass surgery.

OBJECTIVE: To investigate whether ischaemic preconditioning could reduce myocardial injury, as manifest by troponin T release, in patients undergoing elective coronary artery bypass surgery. DESIGN: Randomised controlled trial. SETTING: Cardiothoracic unit of a tertiary care centre. PATIENTS: Patients with three vessel coronary artery disease and stable angina admitted for first time elective coronary artery bypass surgery were invited to take part in the study; 33 patients were randomised into control or preconditioning groups. INTERVENTION: Patients in the preconditioning group were exposed to two additional three minute periods of myocardial ischaemia at the beginning of the revascularisation operation, before the ischaemic period used for the first coronary artery bypass graft distal anastomosis. MAIN OUTCOME MEASURE: Serum troponin T concentration at 72 hours after cardiopulmonary bypass. RESULTS: The troponin T assays were performed by blinded observers at a different hospital. All patients had undetectable serum troponin T (< 0.1 microgram/l) before cardiopulmonary bypass, and troponin T was raised postoperatively in all patients. At 72 hours, serum troponin T was lower (P = 0.05) in the preconditioned group (median 0.3 microgram/l) than in the control group (median 1.4 micrograms/l). CONCLUSIONS: The direct application of a preconditioning stimulus in clinical practice has been shown, for the first time, to protect patients against irreversible myocyte injury.

Characterisation of the infarct-limiting effect of delayed preconditioning: timecourse and dose-dependency studies in rabbit myocardium.

The delayed phase (‘second window’) of protection induced by ischemic preconditioning in rabbit heart is observed as enhanced resilience to infarction 24 hours after repetitive brief cycles of ischemia. Here we provide a fuller physiological characterisation of this phenomenon in the open-chest rabbit model, examining temporal characteristics and dose-dependency of this adaptation. For examination of the timecourse of delayed protection, rabbits were pretreated with four 5 minute coronary artery occlusions (PC) or sham operation (SHAM). Twenty four, 48, 72 or 96 hours later, infarct size after 30 min coronary occlusion and 120 minutes reperfusion was assessed with TTC staining and expressed as a percentage of myocardial risk volume (I/R). I/R was reduced at 24 hours (SHAM 48.1±3.9% v PC31.4±3.0%, P<0.01), 48 hours (SHAM 41.9±3.0% v PC 19.6±6.3%, P<0.01), and 72 hours (SHAM 39.8±3.4% v PC 17.2±2.5%, P<0.01). No protection was observed 96 hours after preconditioning (SHAM 35.0±4.8% v PC 36.9±3.8%). In a further study, animals were pretreated with one, two or four 5 minute coronary occlusions (1×5 PC, 2×5 PC, 4×5 PC) and subjected to the infarction protocol 48 hours later. I/R was 44.5±4.3% in SHAM, 24.8±4.4% in 1×5 PC (P<0.01), 27.4±2.9% in 2×5 PC (P<0.05) and 24.4±4.8 in 4×5 PC (P<0.01). Delayed protection in this rabbit model is prolonged, extending between 24 and 72 hours after the preconditioning stimulus. The threshold for eliciting the second window of protection in this model is as low as one 5 minute coronary occlusion.

Effect of remote ischaemic preconditioning on clinical outcomes in patients undergoing cardiac bypass surgery: a randomised controlled clinical trial

Objectives Remote ischaemic preconditioning (RIPC), using brief cycles of limb ischaemia/reperfusion, is a non-invasive, low-cost intervention that may reduce perioperative myocardial injury (PMI) in patients undergoing cardiac surgery. We investigated whether RIPC can also improve short-term clinical outcomes. Methods One hundred and eighty patients undergoing elective coronary artery bypass graft (CABG) surgery and/or valve surgery were randomised to receive either RIPC (2–5 min cycles of simultaneous upper arm and thigh cuff inflation/deflation; N=90) or control (uninflated cuffs placed on the upper arm and thigh; N=90). The study primary end point was PMI, measured by 72 h area under the curve (AUC) serum high-sensitive troponin-T (hsTnT); secondary end point included short-term clinical outcomes. Results RIPC reduced PMI magnitude by 26% (−9.303 difference (CI −15.618 to −2.987) 72 h hsTnT-AUC; p=0.003) compared with control. There was also evidence that RIPC reduced the incidence of postoperative atrial fibrillation by 54% (11% RIPC vs 24% control; p=0.031) and decreased the incidence of acute kidney injury by 48% (10.0% RIPC vs 21.0% control; p=0.063), and intensive care unit stay by 1 day (2.0 days RIPC (CI 1.0 to 4.0) vs 3.0 days control (CI 2.0 to 4.5); p=0.043). In a post hoc analysis, we found that control patients administered intravenous glyceryl trinitrate (GTN) intraoperatively sustained 39% less PMI compared with those not receiving GTN, and RIPC did not appear to reduce PMI in patients given GTN. Conclusions RIPC reduced the extent of PMI in patients undergoing CABG and/or valve surgery. RIPC may also have beneficial effects on short-term clinical outcomes, although this will need to be confirmed in future studies. Trial registration number ClinicalTrials.gov ID: NCT00397163.

Ischaemic preconditioning may abolish the protection afforded by ATP-sensitive potassium channel openers in isolated human atrial muscle

The ATP-sensitive potassium channel (KATP channel) has been implicated in the mechanism underlying ischaemic preconditioning protection. This study based on human atrium compared the protective effects of ischaemic preconditioning with pre-operative nicorandil (a KATP channel opener with nitrate actions). We also examined the added effect of ischaemic preconditioning to that of nicorandil on ischaemic protection. The protective effects of other KATP channel openers devoid of nitrate actions were also examined.Atrial trabeculae harvested from patients undergoing routine myocardial revascularisation were divided on the basis of whether patients had been ingesting nicorandil orally preoperatively. Trabeculae were superfused with oxygenated Tyrodes solution and following stabilisation underwent 90 minutes simulated ischaemia followed by 120 minutes reoxygenation (n=6 per group). Atrial trabeculae exposed to nicorandil underwent either no treatment (N), or ischaemic preconditioning (N+PC) using 3 minutes simulated ischaemia and 7 minutes reoxygenation prior to the 90 minutes simulated ischaemia. Similarly trabeculae not exposed to nicorandil underwent either no treatment, controls (C), or ischaemic preconditioning (PC). The experimental endpoint was recovery of contractile function presented as percentage baseline function. Further groups were examined using other KATP channels openers with and without ischaemic preconditioning.In the control group, following 120 minutes reoxygentation the recovery of function reached 28.8±3.5%. In contrast, exposure to nicorandil alone improved recovery of function (55.5%±5.3) to a similar extent as PC (55.3%±2.5) when compared to controls (p<0.05, ANOVA). The addition of ischaemic preconditioning to nicorandil exposure abolished protection (29.7%±3.1). Findings were confirmed using the other KATP channels openers.Clinically available nicorandil appears to afford ischaemic protection to isolated human atrial muscle. The addition of a short ischaemic episode to nicorandil exposure seems to completely abolish this protection. Although the mechanism underlying this effect remains unknown, we believe that this observation may have clinical implications.

Delayed myocardial protection following ischaemic preconditioning.

The last two years have brought the realisation that ischaemic preconditioning of myocardium is associated with a biphasic pattern of myocardial protection. In addition to the well-researched early phase of protection (‘classic’ preconditioning) there is now a considerable body of evidence pointing to a much delayed phase of protection which we have termed the ‘second window of protection’ (19). That two distinct phase of protection occur in response to transient ischaemic stress suggests that there are at least two different endogenous routes to cytoprotection in myocardium. Both forms of adaptation may be extremely pertinent to our conceptions of the natural history of ischaemic heart disease and both may provide new templates for the development of cardioprotective strategies. Our own feeling is that the second window of protection is an adaptive cytoprotection sharing many of the features of the cellular stress response, widely observed in prokaryotic and eukaryotic cells, and in several mammalian tissues. Here, we outline the evidence for a second window of protection in myocardium, discuss some current concepts concerning underlying mechanisms, and speculate on its pathophysiological relevance.

PHARMACOLOGICAL PRECONDITIONING OF PRIMARY RAT CARDIAC MYOCYTES BY FK506

Pre-treatment with the immunosuppressant FK506 is shown to protect primary cardiocytes against a subsequent severe thermal or ischaemic stress. This effect is not observed with the related compounds cyclosporin A or rapamycin. It does not involve induction of the FK506 binding, heat inducible protein hsp56 or of the other heat shock proteins. In addition over-expression of hsp56 does not protect cardiac cells from severe stress in contrast to our previous results with hsp70 and hsp90. These results suggest the FK506 is acting via a novel mechanism to protect cardiac cells against cellular ischaemia which may not be related to its immunosuppressant action.

Cardioprotective Properties of the Platelet P2Y12 Receptor Inhibitor, Cangrelor: Protective in Diabetics and Reliant Upon the Presence of Blood.

Dual antiplatelet therapy represents a cornerstone of the current management of acute coronary syndromes. Combining pharmacological anti-platelet agents, the cyclo-oxygenase (COX) inhibitor aspirin, with a purine P2Y12 receptor inhibitor, these pharmacological platelet inhibitors are orally loaded at the time of diagnosis prior to the patient entering the cardiac catheter laboratory. Until recently, the primary physiological and pharmacological focus of these agents has been appropriately directed towards their ability to alter the rheology of the blood; reducing platelet aggregability to attenuate the risk of stent thrombosis. However, there is now an increasing awareness of the pleotropic properties of anti-platelet therapies, particularly of the P2Y12 inhibitors, to ameliorate myocardial ischaemia/reperfusion injury. Recently, it has been demonstrated that administration of P2Y12 inhibitors prior to the onset of reperfusion can result in a significant reduction of infarct size. Interestingly, this appears to be a class-effect, with a range of chemically distinct (thienopyridine and nonthienopyridine) P2Y12 inhibitors demonstrating the same cardioprotective ability to ameliorating infarct size, provided that an adequate circulating concentration of the inhibitor was present at the moment of reperfusion [1]. The critical difference between thienopyridine and non-thienopyridine P2Y12 inhibitors concerns the rate of onset. The first P2Y12 inhibitor to gain widespread acceptance in the management of acute coronary syndromes was the thienopyridine clopidogrel. Clopidogrel, like the other widely used thienopyridine, prasugrel, requires a significant period of time to realise P2Y12 inhibition by virtue of the need for hepatic P450-mediated conversion of the pro-drug into its active metabolite [2]. The requirement for metabolic conversion is not a problem with the non-thienopyridine P2Y12 inhibitors such as Ticagrelor and Cangrelor [2], but the absorption time from the gut—particularly in patients receiving opiate analgesia—will delay the onset of adequate P2Y12 inhibition for drugs with an oral route of administration [3]. With a rapid onset of action and the ability to quickly and reliably load the circulation through intravenous bolus and subsequent infusion, Cangrelor is perhaps the ideal antiplatelet therapy for use in the emergency management of ST-segment elevation myocardial infarction, ensuring rapid and effective platelet inhibition at the time of revascularisation and stent deployment. The hitherto unexpected (and unrealised) potential clinical advantage of Cangrelor is the amelioration of ischaemia/ reperfusion injury. Not currently targeted as part of routine clinical management, the excess myocardial cell death resulting from the restoration of blood and oxygen supply can contribute up to 50 % of the final infarct size in experimental and clinical studies [4]. Ischaemia/reperfusion injury therefore represents a clear and currently unmet clinical need—a need that could be met through re-purposing of a therapeutic intervention that is already in widespread clinical * D. M. Yellon d.yellon@ucl.ac.uk

Protection Against Global Ischemia in the Rabbit Isolated Heart 24 Hours After Transient Adenosine A1 Receptor Activation

Dear Sir, Preconditioning myocardium with brief periods of ischemia renders the heart more resistant to subsequent prolonged ischemia. There is a wealth of evidence that endogenously released adenosine, acting on the adenosine A1 receptor, is an important trigger of classic preconditioning in the rabbit. In the blood-perfused rabbit isolated heart, Liu et al. [1] were able to abolish the infarct-limiting effect of preconditioning when 8-(p-sulphophenyl)theophylline (SPT), a nonselective adenosine receptor antagonist, was administered during preconditioning. Conversely, protection against infarction was elicited by administering the selective adenosine A1 agonist R-phenyl-isopropyladenosine in place of preconditioning. This work was extended to an in vivo rabbit model in which it was shown that 2-chloro-N6cyclopentyladenosine (CCPA), a highly selective adenosine A1 receptor agonist [2], given 10 minutes before 30 minutes of regional ischemia, signi~cantly limited infarct development [3]. It is known that ischemic preconditioning also induces a delayed phase of protection evident around 24–72 hours after the preconditioning stimulus [4]. Studies in our laboratory have suggested that adenosine receptor activation during preconditioning in the rabbit might be an important trigger of not only early protection but also of delayed protection. We showed that adenosine receptor blockade with SPT during preconditioning abolished the protection against infarction 24 hours later [5]. We also demonstrated in the same study that a single bolus of CCPA (25, 50, or 100 lg/kg) led to enhanced resistance to infarction 24 hours later in a rabbit model of coronary occlusion in vivo. We now report a further study that we have undertaken to investigate delayed myocardial protection triggered by transient adenosine A1 receptor activation in the rabbit. We hypothesized that A1 receptor activation evokes protection through a direct action on the myocardium rather than through humoral, neuronal, or blood-borne factors. To test this hypothesis, responses to ischemiareperfusion were examined in the rabbit isolated heart 24 hours after CCPA treatment. Male New Zealand White rabbits (2.0–3.0 kg) received a single bolus of CCPA (100 lg/kg in 0.9% NaCl 1.0 ml; Research Biochemicals, Natick, MA, USA) or saline vehicle by intravenous injection through a marginal ear vein. The animals were immediately returned to their pens with no further manipulation. Twentyfour hours after pretreatment, rabbits were anesthetized with pentobarbitone sodium, and after systemic heparinization (500 IU i.v.) the hearts were excised and retrograde perfused in the nonrecirculating Langendorff mode with Krebs-Henseleit buffer of the following composition (mmol/l): NaCl 118, NaHCO3 25, KCl 4.2, KH2PO4 1.8, CaCl2 1.8, MgSO4 1.2, and d-glucose 10 (pH 7.35–7.45 when equilibrated with 95% O2/5% CO2). Hearts were perfused at a constant _ow rate of 35 ml/min. A _uid-~lled latex balloon was introduced into the left ventricle, and the volume was adjusted to give an end-diastolic pressure 5–10 mmHg. This preload was maintained throughout the experiment. After stabilization, global ischemia was induced by stopping aortic _ow. During 30 minute global ischemia the temperature was maintained at 37.0 6 0.58C and the hearts were paced at 3 Hz. Following the period of global ischemia, _ow was re-instated at the preischemic value for 60 minutes, during which time left ventricular end-diastolic pressure and left ventricular developed pressure were monitored. Samples of coronary effluent were collected at the end of stabilization (baseline) and at 15-minute intervals dur-

Pre-conditioning with adenosine leads to concentration-dependent infarct size reduction in the isolated rabbit heart

Adenosine (ADO) has a cardioprotective effect in ischemia-reperfusion injury when administered both prior to ischemia and during reperfusion. ADO has also been implicated in the mechanism of ischemic pre-conditioning. The aim of this study was to investigate whether there was a concentration-response between the administration of ADO prior to ischemia-reperfusion and reduction in subsequent infarct size. Rabbit isolated perfused hearts were subjected to 45 min ischemia and 180 min reperfusion following pre-treatment with either Krebs Henseleit buffer alone or buffer containing ADO at a range of concentrations (3 micro M-100 micro M) for 5 min followed by 5 min perfusion with buffer. Infarct/risk ratios were significantly reduced in hearts pre-perfused with higher (> 3 micro M) concentrations of ADO (Control, 58.5 +/- 1.5%; 3 micro M ADO, 51.6 +/- 3.0% ; 6 micro M ADO, 44.1% +/- 2.0%; 10 micro M ADO, 33.3 +/- 1.9%; 20 micro M ADO, 26.6 +/- 0.9%; 50 micro M ADO, 21.6 +/- 3.5%; 100 micro M ADO, 23.0 +/- 0.6%). We conclude that pre-treatment with ADO leads to a concentration-dependent reduction in infarct size.