Xi-Ming Yang

Role of Bradykinin in Protection of Ischemic Preconditioning in Rabbit Hearts

Bradykinin receptor activation has been proposed to be involved in ischemic preconditioning. In the present study, we further investigated the role of this agent in preconditioning in both isolated and in situ rabbit hearts. All hearts were subjected to 30 minutes of regional ischemia followed by reperfusion for 2 hours (in vitro hearts) and 3 hours (in situ hearts). Infarct size was measured by tetrazolium staining and expressed as a percentage of the size of the risk zone. Preconditioning in situ hearts with 5 minutes of ischemia and 10 minutes of reperfusion significantly reduced infarct size to 10.2 +/- 2.2% of the risk region (P < .0005 versus control infarct size of 36.7 +/- 2.6%). Pretreatment with HOE 140 (26 micrograms/kg), a bradykinin B2 receptor blocker, did not alter infarct size in nonpreconditioned hearts (40.6 +/- 5.3% infarction) but abolished protection from ischemic preconditioning (34.1 +/- 1.6% infarction). However, when HOE 140 was administered during the initial reflow period following 5 minutes of ischemia, protection was no longer abolished (15.6 +/- 3.9% infarction versus 13.3 +/- 3.8% without HOE 140, P = NS). Bradykinin infusion in isolated hearts mimicked preconditioning, and protection was not affected by pretreatment with the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester or the prostaglandin synthesis inhibitor indomethacin but could be completely abolished by the protein kinase C (PKC) inhibitors polymyxin B and staurosporine as well as by HOE 140. HOE 140 could not block the protection of ischemic preconditioning in isolated hearts. That failure was apparently due to the absence of blood-borne kininogens rather than autonomic nerves. When the preconditioning stimulus in the in situ model was amplified with four cycles of 5-minute ischemia/10-minute reperfusion, HOE 140 pretreatment could no longer block protection (infarct size was 10.7 +/- 3.5% versus 6.4 +/- 2.0% without HOE 140, P = NS). We propose that bradykinin receptors protect by coupling to PKC as do adenosine receptors, and blockade of either receptor will diminish the total stimulus of PKC below threshold and prevent protection. A more intense preconditioning ischemic stimulus can overcome bradykinin receptor blockade, however, by simply enhancing the amount of adenosine and possibly other agonists released.

The pH Hypothesis of Postconditioning Staccato Reperfusion Reintroduces Oxygen and Perpetuates Myocardial Acidosis

Background— It is unclear how reperfusion of infarcting hearts with alternating cycles of coronary reperfusion/occlusion attenuates infarction, but prevention of mitochondrial permeability transition pore (MPTP) formation is crucial. Acidosis also suppresses MPTP formation. We tested whether postconditioning protects by maintaining acidosis during early reoxygenation. Methods and Results— After 30-minute regional ischemia in isolated rabbit hearts, reperfusion with buffer (pH 7.4) caused 34.4±2.2% of the risk zone to infarct, whereas 2 minutes of postconditioning (6 cycles of 10-second reperfusion/10-second occlusion) at reperfusion resulted in 10.7±2.9% infarction. One minute (3 cycles) of postconditioning was not protective. Hypercapnic buffer (pH 6.9) for the first 2 minutes of reperfusion in lieu of postconditioning caused equivalent cardioprotection (15.0±2.6% infarction), whereas 1 minute of acidosis did not protect. Delaying postconditioning (6 cycles) or 2 minutes of acidosis for 1 minute aborted protection. Reperfusion with buffer (pH 7.7) blocked postconditioning protection, but addition of the MPTP closer cyclosporin A restored protection. Reactive oxygen species scavenger N-2-mercaptopropionyl glycine, protein kinase C antagonist chelerythrine, and mitochondrial KATP channel closer 5-hydroxydecanoate each blocked protection from 2 minutes of acidosis as they did for postconditioning. Conclusion— Thus, postconditioning prevents MPTP formation by maintaining acidosis during the first minutes of reperfusion as reoxygenated myocardium produces reactive oxygen species that activate protective signaling to inhibit MPTP formation after pH normalization.

Postconditioning’s protection is not dependent on circulating blood factors or cells but involves adenosine receptors and requires PI3–kinase and guanylyl cyclase activation

Abstract Protection from postconditioning has been documented in in situ animal models and it has been proposed that it is targeting circulating leukocytes. We therefore tested whether postconditioning can protect leukocytefree, buffer–perfused rabbit hearts. Infarct size was measured with triphenyltetrazolium staining. In control hearts undergoing 30 min of regional ischemia and 2 h of reperfusion, 33.3 ± 2.2% of the risk zone infarcted. The protocol previously used in open–chest animals of four postconditioning cycles of 30 s reperfusion/30 s ischemia starting at the beginning of reperfusion decreased infarction to only 24.8 ± 2.5% of the risk zone in these isolated hearts. Because of the meager protection induced by four 30 s postconditioning cycles, we evaluated the effect of postconditioning with 6 cycles of 10 s reperfusion/10 s ischemia starting at the beginning of reperfusion. Robust salvage was seen with only 10.4 ± 3.4% of the risk zone infarcting (p < 0.001 vs control and p < 0.003 vs 4 cycles of 30 s ischemia). The 10s protocol was used in all studies of signal transduction. Wortmannin (100 nM), a phosphatidylinositol 3– (PI3–) kinase antagonist, infused for 20 min starting 5 min before reperfusion, blocked postconditioning’s, protection (31.2 ± 4.2% infarction) as did 1H–[1,2,4]oxadiazole[4,3–a]quinoxalin–1–one (ODQ) (2 µM) a guanylyl cyclase inhibitor (36.9 ± 5.3%) and 8–p–(sulfophenyl) theophylline (SPT) (100 µM), a non–specific adenosine receptor blocker (34.2 ± 2.8%). Thus, postconditioning’s protection is not dependent on circulating blood factors or cells, and its anti–infarct effect appears to require PI3–kinase activation, stimulation of guanylyl cyclase and occupancy of adenosine receptors. These signaling steps have also been identified in preconditioning and during pharmacologic cardioprotection and suggest commonality of a protective mechanism.

Conscious rabbits become tolerant to multiple episodes of ischemic preconditioning.

Although ischemic preconditioning protects myocardium from infarction in isolated hearts and in anesthetized open-chest animals, its effects have not been examined in unanesthetized animals. Furthermore, it is unknown whether animals become tolerant to multiple episodes of ischemic preconditioning. Rabbits were chronically instrumented with a balloon occluder around a major branch of the left coronary artery for reversible coronary occlusion, a left atrial catheter for radioactive microsphere injections, ECG electrodes for monitoring of myocardial ischemia, and, in some cases, a carotid artery catheter for pressure measurements and timed withdrawal of reference arterial blood samples. Eight control rabbits underwent a 30-minute coronary occlusion and then 180 minutes of reperfusion. Five of the eight rabbits developed ventricular tachycardia or fibrillation during ischemia, and infarct size averaged 37.7 +/- 2.6% of the risk area. Eight rabbits experienced a 5-minute coronary occlusion and 10 minutes of reperfusion before the 30-minute occlusion. In these preconditioned animals, potentially fatal arrhythmias during ischemia were significantly reduced (one of eight, P < .05), and infarct size was much smaller (5.6 +/- 1.1%, P < .0001). The difference could not be explained by hemodynamics or collateral blood flow, which were nearly identical in the two groups. But when the 30-minute coronary occlusion was preceded by 40 to 65 five-minute occlusions during a 3- to 4-day period in seven animals, protection was markedly attenuated. Potentially lethal arrhythmias were very common, and infarct size averaged 26.5 +/- 2.9%, substantially larger than in rabbits with only one preconditioning occlusion (P < .0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Infarct limitation of the second window of protection in a conscious rabbit model

OBJECTIVES Myocardial protection associated with ischemic preconditioning (PC) wanes within an hour or two. It has recently been observed, however, that a delayed phase of protection appears about 24 h after ischemic PC in anesthetized rabbits and dogs which might be related to synthesis of cytoprotective proteins. We tested whether a second window of protection could be induced in conscious rabbits. METHODS Rabbits chronically instrumented with a coronary artery occluder and ECG electrodes experienced a 30-min coronary occlusion followed by 3 h reperfusion. Infarct size was measured with triphenyltetrazolium chloride. RESULTS 35.7 +/- 2.3% of the risk zone infarcted in control animals. PC with 4 cycles of 5-min coronary occlusion/10-min reperfusion 24 h prior to the 30-min ischemia decreased infarction to 24.1 +/- 1.4% of the risk zone (P < 0.01). During the 30-min occlusion 3 of 7 non-PC rabbits developed ventricular fibrillation, while this arrhythmia did not occur in the 7 PC animals (P < 0.1). Myocardial hsp70 content in PC rabbits was twice that in controls. Collateral blood flow was not different in the two groups. CONCLUSIONS A second window of protection exists in conscious rabbits which minimizes both infarction and arrhythmias, and cytoprotective protein content is increased in the myocardium of protected animals.

Catecholamines can induce adenosine receptor-mediated protection of the myocardium but do not participate in ischemic preconditioning in the rabbit.

The role of catecholamines in ischemic preconditioning is unclear. Accordingly, the effects of tyramine-induced norepinephrine release and alpha 1-receptor blockade were examined. Ischemic preconditioning with a 5-minute coronary occlusion 10 minutes before a 30-minute ischemic interval resulted in only 7.7 +/- 3.1% infarction of the risk area, significantly less than that in control rabbits with isolated 30-minute coronary occlusions (34.4 +/- 3.2%, P < .01). Intravenous infusion of tyramine 10 minutes before 30 minutes of ischemia also protected the heart from infarction to an extent similar to that seen with ischemic preconditioning (6.9 +/- 2.4% infarction). This protection observed with tyramine infusion was eliminated by alpha 1-receptor blockade with BE 2254 (36.8 +/- 2.6% infarction) but was unaffected by beta-blockade with propranolol (10.5 +/- 2.4% infarction). Furthermore, the protection was unaffected when the tyramine-induced hypertension was attenuated by allowing blood to flow into a volume reservoir (3.9 +/- 0.8% infarction, P < .01 vs control value). The nonselective adenosine-receptor blocker PD 115,199 also eliminated tyramine-induced protection (40.2 +/- 5.6% infarction), indicating that adenosine is involved in adrenergic-mediated protection. BE 2254 could not block ischemic preconditioning (3.9 +/- 1.1% infarction, P < .01 vs control value). Therefore, catecholamine release before prolonged ischemia can protect the heart from infarction via the alpha 1-receptor, but adenosine receptor stimulation is also involved. alpha-Adrenergic stimulation does not appear to be critical to the protection observed after ischemic preconditioning.

Platelet P2Y12 Blockers Confer Direct Postconditioning-Like Protection in Reperfused Rabbit Hearts

Background: Blockade of platelet activation during primary percutaneous intervention for acute myocardial infarction is standard care to minimize stent thrombosis. To determine whether antiplatelet agents offer any direct cardioprotective effect, we tested whether they could modify infarction in a rabbit model of ischemia/reperfusion caused by reversible ligation of a coronary artery. Methods and Results: The P2Y12 (adenosine diphosphate) receptor blocker cangrelor administered shortly before reperfusion in rabbits undergoing 30-minute regional ischemia/3-hour reperfusion reduced infarction from 38% of ischemic zone in control hearts to only 19%. Protection was dose dependent and correlated with the degree of inhibition of platelet aggregation. Protection was comparable to that seen with ischemic postconditioning (IPOC). Cangrelor protection, but not its inhibition of platelet aggregation, was abolished by the same signaling inhibitors that block protection from IPOC suggesting protection resulted from protective signaling rather than anticoagulation. As with IPOC, protection was lost when cangrelor administration was delayed until 10 minutes after reperfusion and no added protection was seen when cangrelor and IPOC were combined. These findings suggest both IPOC and cangrelor may protect by the same mechanism. No protection was seen when cangrelor was used in crystalloid-perfused isolated hearts indicating some component in whole blood is required for protection. Clopidogrel had a very slow onset of action requiring 2 days of treatment before platelets were inhibited, and only then the hearts were protected. Signaling inhibitors given just prior to reperfusion blocked clopidogrel’s protection. Neither aspirin nor heparin was protective. Conclusions: Clopidogrel and cangrelor protected rabbit hearts against infarction. The mechanism appears to involve signal transduction during reperfusion rather than inhibition of intravascular coagulation. We hypothesize that both drugs protect by activating IPOC’s protective signaling to prevent reperfusion injury. If true, patients receiving P2Y12 inhibitors before percutaneous intervention may already be postconditioned thus explaining failure of recent clinical trials of postconditioning drugs.

Acadesine extends the window of protection afforded by ischaemic preconditioning in conscious rabbits

OBJECTIVE Ischaemic preconditioning protects myocardium from infarction if the reperfusion interval between the brief and prolonged ischaemic intervals is less than 1 h. In anaesthetised rabbits acadesine (5-amino-4-imidazolecarboxamide riboside, AICAR), an adenosine enhancer which increases tissue adenosine during ischaemia, prolongs the window of protection to 2 h. The aim of this study was to try to determine the maximum extension of this window of protection, using chronically instrumented, unsedated rabbits. METHODS Rabbits were instrumented with a balloon occluder around a major branch of the left coronary artery for reversible coronary occlusion. Five to seven days after surgery all animals underwent a 30 min coronary occlusion. Animals were randomised to one of seven groups: (1) No additional treatment (control); (2) Ischaemic preconditioning with 5 min regional ischaemia followed by 10 min reperfusion before the 30 min coronary occlusion; (3) and (4) Ischaemic preconditioning followed by 2 or 4 h of reperfusion before the 30 min occlusion, respectively; (5) Treatment with acadesine (2.5 mg.kg-1.min-1 intravenously for 5 min and then 0.5 mg.kg-1.min-1 beginning 45 min before and continuing until 30 min after release of the 30 min occlusion) without ischaemic preconditioning; (6) and (7) Treatment with the higher dose of acadesine for 5 min beginning 35 min before the 5 min ischaemic period, and then the lower dose continuing until 30 min after release of the 30 min coronary occlusion in rabbits with 4 or 6 h reperfusion intervals, respectively. RESULTS Rabbits with ischaemic preconditioning with 10 min reperfusion preceding the 30 min coronary occlusion (group 2) had only 5.6(SEM 1.1)% infarction of the ischaemic zone. Ischaemic preconditioning followed by 2 h reperfusion (group 3) offered continued protection [18.2(2.2)% infarction] as compared to control animals [37.7(2.6)% infarction]. However, protection waned if ischaemic preconditioning was followed by 4 h reperfusion (group 4) [36.7(3.0)% infarction]. Additionally, treatment with acadesine alone did not modify infarct size (group 7) [39.5(4.0)%], but acadesine largely restored the protection of ischaemic preconditioning despite a 4 h reperfusion interval (group 5) [20.4(3.0)% infarction, P < 0.01 v control]. However, when reperfusion was extended to 6 h (group 6) acadesine could no longer restore protection [36.2(0.9)% infarction]. CONCLUSIONS The protection afforded by a 5 min ischaemic preconditioning period lasts from 2 to 4 h in the awake, unsedated rabbit, and acadesine can extend the duration of this window of protection to at least 4 h but not to 6 h.

Smaller infarct after preconditioning does not predict extent of early functional improvement of reperfused heart

We evaluated the ability of ischemic preconditioning to restore function to salvaged myocardium in rabbits. Although ischemic preconditioning reduces infarct size, few investigators studying recovery of function after coronary occlusions lasting ≥30 min have reported any mechanical benefit in preconditioned hearts. However, because myocardial function was seldom evaluated beyond 5 h after reperfusion stunning may have masked the benefit. Accordingly, rabbits were chronically instrumented with a pneumatic occluder around a branch of the left coronary artery, a pair of 1-mm ultrasonic crystals in the myocardial territory destined to become ischemic, and electrocardiogram (ECG) leads. One week after surgery the ECG and segment length tracing were recorded at rest, during 30-min occlusion and 1 h of reflow, and again at 24, 48, and 72 h. In ischemically preconditioned rabbits, 5-min coronary occlusion and 10-min reperfusion preceded the long occlusion. The beginning and end of systole were determined by recording the first and second heart sounds with a hand-held precordial microphone. Postmortem infarct size was measured with triphenyltetrazolium chloride. During the 30-min coronary occlusion all segments became nearly akinetic or bulged during systole. After 60 min of reflow there was little return of function in either group. Between 24 and 72 h there was minimal recovery in the control group (segment shortening equals 13.3 ± 4.1% of baseline), whereas function was much better in preconditioned hearts (44.2 ± 7.4% of baseline, P < 0.02). Infarct size as a percentage of risk zone was much smaller in preconditioned hearts (10.2 ± 1.4 vs. 29.7 ± 1.8%, P < 0.001). Thus there is a gradual recovery of systolic function of reperfused myocardium after a coronary occlusion. Although early mechanical recovery is significantly better after preconditioning, it is much less than would be predicted by the reduction of infarct size.We evaluated the ability of ischemic preconditioning to restore function to salvaged myocardium in rabbits. Although ischemic preconditioning reduces infarct size, few investigators studying recovery of function after coronary occlusions lasting >/=30 min have reported any mechanical benefit in preconditioned hearts. However, because myocardial function was seldom evaluated beyond 5 h after reperfusion stunning may have masked the benefit. Accordingly, rabbits were chronically instrumented with a pneumatic occluder around a branch of the left coronary artery, a pair of 1-mm ultrasonic crystals in the myocardial territory destined to become ischemic, and electrocardiogram (ECG) leads. One week after surgery the ECG and segment length tracing were recorded at rest, during 30-min occlusion and 1 h of reflow, and again at 24, 48, and 72 h. In ischemically preconditioned rabbits, 5-min coronary occlusion and 10-min reperfusion preceded the long occlusion. The beginning and end of systole were determined by recording the first and second heart sounds with a hand-held precordial microphone. Postmortem infarct size was measured with triphenyltetrazolium chloride. During the 30-min coronary occlusion all segments became nearly akinetic or bulged during systole. After 60 min of reflow there was little return of function in either group. Between 24 and 72 h there was minimal recovery in the control group (segment shortening equals 13.3 +/- 4.1% of baseline), whereas function was much better in preconditioned hearts (44.2 +/- 7.4% of baseline, P < 0.02). Infarct size as a percentage of risk zone was much smaller in preconditioned hearts (10.2 +/- 1.4 vs. 29.7 +/- 1.8%, P < 0.001). Thus there is a gradual recovery of systolic function of reperfused myocardium after a coronary occlusion. Although early mechanical recovery is significantly better after preconditioning, it is much less than would be predicted by the reduction of infarct size.

Favorable Remodeling Enhances Recovery of Regional Myocardial Function in the Weeks After Infarction in Ischemically Preconditioned Hearts

BACKGROUND In a previous study, we found that recovery of segment shortening in the ischemic zone of conscious, chronically instrumented rabbits was significantly better in ischemically preconditioned than control animals after 72 hours of reperfusion. However, although this period of reperfusion was felt to be sufficient to allow recovery from stunning, regional function was disproportionately low for the size of the infarcts. METHODS AND RESULTS To further characterize the recovery of left ventricular regional function, rabbits were chronically instrumented with a balloon occluder around a branch of the left coronary artery and a pair of ultrasonic crystals to monitor segment shortening in the ischemic zone. The preconditioned group had 1 cycle of 5-minute occlusion/10-minute reperfusion before a 30-minute occlusion, whereas control rabbits experienced only the 30-minute occlusion. All monitored segments were either dyskinetic or akinetic during the 30-minute occlusion. There was no difference in function between the 2 groups until 24 hours of reperfusion. At 72 hours, systolic shortening in control hearts averaged only 5% of the preischemic value, whereas shortening was 29% of baseline in preconditioned hearts. By day 21, systolic shortening averaged 26% in control hearts and 65% in preconditioned hearts (P<0.02) and appeared to have reached a plateau. Infarct size was 31.4+/-2.8% and 15.5+/-2.1% in control and preconditioned hearts, respectively. Moreover, in ischemically preconditioned hearts, the recovery of regional function was better than in controls for any given amount of microinfarction in the myocardial segment between crystals (P=0.02). CONCLUSIONS The progressive improvement in preconditioned hearts is most consistent with favorable remodeling in the ischemic zone, which the preconditioning process seems to accentuate.