Adenosine-enhanced ischemic preconditioning provides enhanced postischemic recovery and limitation of infarct size in the rabbit heart

OBJECTIVE: The purpose of this study was to determine the effect of an intracoronary bolus injection of adenosine used in concert with ischemic preconditioning on postischemic functional recovery and infarct size reduction in the rabbit heart and to compare adenosine-enhanced ischemic preconditioning with ischemic preconditioning and magnesium-supplemented potassium cardioplegia. METHODS: New Zealand White rabbits (n = 36) were used for Langendorff perfusion. Control hearts were perfused at 37 degrees C for 180 minutes; global ischemic hearts received 30 minutes of global ischemia and 120 minutes of reperfusion; magnesium-supplemented potassium cardioplegic hearts received cardioplegia 5 minutes before global ischemia; ischemic preconditioned hearts received 5 minutes of zero-flow global ischemia and 5 minutes of reperfusion before global ischemia; adenosine-enhanced ischemic preconditioned hearts received a bolus injection of adenosine just before the preconditioning. To separate the effects of adenosine from adenosine-enhanced ischemic preconditioning, a control group received a bolus injection of adenosine 10 minutes before global ischemia. RESULTS: Infarct volume in global ischemic hearts was 32.9% +/- 5.1% and 1.03% +/- 0.3% in control hearts. The infarct volume decreased (10.23% +/- 2.6% and 7.0% +/- 1.6%, respectively; p < 0.001 versus global ischemia) in the ischemic preconditioned group and control group, but this did not enhance postischemic functional recovery. Magnesium-supplemented potassium cardioplegia and adenosine-enhanced ischemic preconditioning significantly decreased infarct volume (2.9% +/- 0.8% and 2.8% +/- 0.55%, respectively; p < 0.001 versus global ischemia, p = 0.02 versus ischemic preconditioning and p = 0.05 versus control group) and significantly enhanced postischemic functional recovery. CONCLUSIONS: Adenosine-enhanced ischemic preconditioning is superior to ischemic preconditioning and provides equal protection to that afforded by magnesium-supplemented potassium cardioplegia.     

Dichotomy of ischemic preconditioning: improved postischemic contractile function despite intensification of ischemic contracture

BACKGROUND: Acceleration of ischemic contracture is conventionally accepted as a predictor of poor postischemic function. Hence, protective interventions such as cardioplegia delay ischemic contracture and improve postischemic contractile recovery. We compared the effect of ischemic preconditioning and cardioplegia (alone and in combination) on ischemic contracture and postischemic contractile recovery. METHODS AND RESULTS: Isolated rat hearts were aerobically perfused with blood for 20 minutes before being subjected to zero-flow normothermic global ischemia for 35 minutes and reperfusion for 40 minutes. Hearts were perfused at a constant pressure for 60 mm Hg and were paced at 360 beats per minute. Left ventricular developed pressure and ischemic contracture were assessed with an intraventricular balloon. Four groups (n=8 hearts per group) were studied: control hearts with 35 minutes of unprotected ischemia, hearts preconditioned with one cycle of 3 minutes of ischemia plus 3 minutes of reperfusion before 35 minutes of ischemia, hearts subjected to cardioplegia with St Thomas' solution infused for 1 minute before 35 minutes of ischemia, and hearts subjected to preconditioning plus cardioplegia before 35 minutes of ischemia. After 40 minutes of reperfusion, each intervention produced a similar improvement in postischemic left ventricular development pressure (expressed as a percentage of its preischemic value: preconditioning, 44 +/- 2%; cardioplegia, 53 +/- 3%; preconditioning plus cardioplegia, 54 +/- 4% and control, 26 +/- 6%, P<.05). However, preconditioning accelerated whereas cardioplegia delayed ischemic contracture; preconditioning plus cardioplegia gave an intermediate result. Thus, times to 75% contracture were as follows: control, 14.3 +/- 0.4 minutes; preconditioning, 6.2 +/- 0.3 minutes; cardioplegia 23.9 +/- 0.8 minutes; and preconditioning plus cardioplegia 15.4 +/- 2.4 minutes (P<.05 preconditioning and cardioplegia versus control). In additional experiments, using blood- and crystalloid-perfused hearts, we describe the relationship between the number of preconditioning cycles and ischemic contracture. CONCLUSIONS: Although preconditioning accelerates, cardioplegia delays, and preconditioning plus cardioplegia has little effect on ischemic contracture, each affords similar protection of postischemic contractile function. These results question the utility of ischemic contracture as a predictor of the protective efficacy of anti-ischemic interventions. They also suggest that preconditioning and cardioplegia may act through very different mechanisms.     

Ischemic preconditioning in the aged heart--myocardial protective effect as compared with the mature heart

It is now well established that pre-treatment with sublethal ischemia, followed by reperfusion, will delay myocardial necrosis during a later sustained ischemic episode, termed ischemic preconditioning (IPC); this has been confirmed experimentally and clinically. However, the effects for the senescent heart differ from those of the mature heart at both functional and cellular levels which have not yet been determined. Comparisons were made between aged (> 135 weeks, n = 18) and mature (15 approximately 20 weeks, n = 8) rabbit hearts which underwent 30 min. normothermic global ischemia with 120 min reperfusion in a buffer-perfused isolated, paced heart model, and the effects of IPC on post-ischemic functional recovery and infarct size were investigated. Ischemic preconditioned hearts (n = 6) were subjected to one cycle of 5 min. global ischemia and 5 min. reperfusion prior to global ischemia. Global ischemic hearts (n = 6) were subjected to 30 min. global ischemia without intervention. Control hearts (n = 6) were subjected to perfusion without ischemia. Post-ischemic functional recovery was better in the ischemic preconditioned hearts than in the global ischemic hearts in both aged and mature hearts. However, in the aged hearts, post-ischemic functional recovery was slightly reduced compared to that of the mature hearts, and only the coronary flow was well-preserved. In the mature hearts, myocardial infarction in the ischemic preconditioned hearts (14.9 +/- 1.3%) and in the control hearts (1.0 +/- 0.3%) was significantly decreased (p < 0.01) compared to that of the global ischemic hearts (32.9 +/- 5.1%). In the aged hearts, myocardial infarction in the ischemic preconditioned hearts (18.9 +/- 2.7%) and in the control hearts (1.1 +/- 0.6%) was significantly decreased (p < 0.001) compared to that of the global ischemic hearts (37.6 +/- 3.7%). The relationship between infarct size and post-ischemic functional recovery of left ventricularpeak developed pressure (LVDP) was linear and the correlation negative, with r = -0.934 (p < 0.001) and -0.875 (p < 0.001) for mature and aged hearts respectively. The data suggest that, in the senescent myocardium, the cellular pathways involved ischemic preconditioning responses that were post-ischemic, and that functional recovery was worse as compared to that of the mature myocardium. Furthermore, the effects of post-ischemic functional recovery became consistently weaker during the control period of 120 min. reperfusion after a prolonged ischemic insult in a buffer perfused isolated rabbit model. However, the effects of infarct size limitation were well-preserved in both senescent and mature myocardia.     

Role of preischemic glycogen depletion in the improvement of postischemic metabolic and contractile recovery of ischemia-preconditioned rat hearts

BACKGROUND: Ischemic preconditioning (IPC) attenuates acidosis during prolonged ischemia and improves contractile and metabolic parameters during subsequent reperfusion. Glycogen depletion induced by IPC is proposed as a potential mechanism. METHODS AND RESULTS: We studied the influence of manipulations of preischemic glycogen levels (Pre-G, micromol glucose/g wet wt) on contractile and metabolic (via 31P-nuclear magnetic resonance) parameters during 30 minutes of ischemia and recovery in four groups of isovolumic rat hearts: First, control (Con, n=18, mean Pre-G, 21.5+/-0.8); second, after two 5-minute IPC periods (IPC, n=12, Pre-G, 11.3+/-0.7); third, a control group in which Pre-G was depleted by glucose-free, acetate perfusion (Con-LowG, n=9, Pre-G, 7.9+/-1.2); and fourth, an IPC group in which Pre-G was raised by glucose and lactate perfusion such that Pre-G was similar to Con (IPC-HiG, n=11, Pre-G, 20+/-1.4). Manipulation of Pre-G significantly altered the pH fall during 30 minutes of ischemia (Con, 5.76+/-.03, Con-LowG, 6.26+/-.07; IPC-HiG, 5.91+/-.02, IPC, 6.05+/-.09). IPC-HiG hearts had significantly worse metabolic recovery (PCr, 70+/-7 versus 91+/-3% initial; IPC-HiG versus IPC, P<.05) and contractile recovery (end-diastolic pressure, 52+/-5 versus 29+/-5 mm Hg, P<.05) than IPC hearts but better recovery than Con (%PCr, 56+/-6% and end-diastolic pressure, 72+/-6 mm Hg). An ischemic rise in intracellular magnesium occurred and was atttenuated in preconditioned hearts. CONCLUSIONS: Pre-G levels before ischemia influence but are not the sole determinants of the extent of acidosis during prolonged ischemia and of metabolic and contractile recovery during reperfusion in control and preconditioned hearts.     

Alterations in pulmonary surfactant composition and activity after experimental lung transplantation

BACKGROUND: Adenosine has been reported to mediate the necrosis-limiting effects of ischemic preconditioning; however, it is unclear how this mediation occurs. The present study was undertaken to test the hypothesis that ischemic preconditioning increases 5'-nucleotidase activity and adenosine release during sustained ischemia and subsequent reperfusion. METHODS AND RESULTS: After thoracotomy, the left anterior descending coronary artery was cannulated and perfused with blood redirected from the left carotid artery in 32 dogs. Ischemic preconditioning was produced by four cycles in which the coronary artery was occluded and then reperfused for 5 minutes each. After the last cycle of ischemia and reperfusion, the coronary artery was occluded for 40 minutes. This was followed by 120 minutes of reperfusion. In the control group, the coronary artery was occluded for 40 minutes and reperfused for 120 minutes without ischemic preconditioning. The plasma adenosine concentration was measured and blood gases were analyzed in coronary arterial and venous blood samples taken during 120 minutes of reperfusion. Myocardial 5'-nucleotidase activity was measured before and at 40 minutes of sustained ischemia with and without ischemic preconditioning. The adenosine concentration in coronary venous blood during reperfusion was significantly higher in preconditioned hearts than in the control hearts: 1 minute after the onset of reperfusion, 546 +/- 57 versus 244 +/- 41 pmol/ml; 10 minutes after, 308 +/- 30 versus 114 +/- 14 pmol/ml; 30 minutes after, 175 +/- 24 versus 82 +/- 16 pmol/ml, respectively (p < 0.01). Ectosolic and cytosolic 5'-nucleotidase activities increased in both endocardial and epicardial myocardium in the ischemia-preconditioned hearts. Furthermore, 40 minutes of ischemia increased 5'-nucleotidase activity in ischemia-preconditioned hearts more than in control hearts. CONCLUSIONS: Ischemic preconditioning increases adenosine release and 5'-nucleotidase activity during sustained ischemia and subsequent reperfusion.     

Screening a peptidyl database for potential ligands to proteins with side-chain flexibility

OBJECTIVE: Microdialysis and 31P-NMR spectroscopy were used to test opposing hypotheses that ischemic preconditioning inhibits adenine nucleotide degradation and purine efflux, or that preconditioning activates cardiovascular adenosine formation to provide enhanced cardioprotection. METHODS: 31P-NMR spectra and matching interstitial fluid (ISF) or venous effluent samples were obtained from Langendorff perfused rat hearts. Control hearts (n = 9) underwent 30 min of global normothermic ischemia and 30 min reperfusion. Preconditioned hearts (n = 6) were subjected to a 5 min ischemic episode and 10 min reflow prior to 30 min ischemia and 30 min reperfusion. Effects of repetitive ischemia-reperfusion (3 x 5 min ischemic episodes) on adenosine levels and energy metabolism were also assessed (n = 8). RESULTS: Preconditioning improved post-ischemic recovery of heart rate x left ventricular developed pressure (71 +/- 5 vs 43 +/- 8%, P < 0.05) and end-diastolic pressure (14 +/- 3 vs 29 +/- 4 mmHg, P < 0.05) compared with control hearts, respectively. Preconditioning did not alter intracellular ATP, phosphocreatine (PCr), inorganic phosphate (Pi), H+ or free Mg2+ during global ischemia, but improved recoveries of PCr, Pi, and delta GATP on reperfusion. ISF adenosine increased more than 20-fold during 30 min ischemia. The 5 min preconditioning episode increased ISF adenosine 3-fold, and reduced ISF adenosine and inosine during subsequent prolonged ischemia by up to 75%. Venous purine levels during reperfusion were also reduced by preconditioning. Accumulation of adenosine in ISF and venous effluent during repetitive ischemia was progressively reduced despite comparable changes in substrate for adenosine formation via 5'-nucleotidase, (5'-AMP), and in allosteric modulators of this enzyme (Mg2+, H+, Pi, ADP, ATP). CONCLUSIONS: (i) Ischemic preconditioning reduces interstitial and vascular adenosine levels during ischemia-reperfusion, (ii) reduced ISF adenosine during ischemia is not due to reduced ischemic depletion of adenine nucleotides in preconditioned rat hearts, (iii) preconditioning may inhibit adenosine formation via 5'-nucleotidase in ischemic rat hearts, and (iv) improved functional recovery with preconditioning is unrelated to metabolic/bioenergetic changes during the ischemic insult, but may be related to improved post-ischemic recovery of [Pi] and delta GATP in this model.     

Ischemic preconditioning attenuates postischemic coronary artery endothelial dysfunction in a model of minimally invasive direct coronary artery bypass grafting

OBJECTIVE: Unmodified reperfusion without cardioplegia in minimally invasive direct coronary artery bypass grafting procedures causes endothelial dysfunction that may predispose to polymorphonuclear neutrophil-mediated myocardial injury. This study tested the hypothesis that ischemic preconditioning in a minimally invasive direct coronary artery bypass grafting model attenuates postischemic endothelial dysfunction in coronary vessels. METHODS: In anesthetized dogs, the left anterior descending coronary artery was occluded for 30 minutes and reperfused for 3 hours without ischemic preconditioning (no-ischemic preconditioning; n = 7); in 7 dogs, the left anterior descending occlusion was preceded by 5 minutes occlusion followed by 5 minutes of reperfusion. Relaxation responses to stimulators of nitric oxide synthase were used to evaluate endothelial function in arteries from the ischemic-reperfused (left anterior descending) and nonischemic (left circumflex coronary artery) zones. RESULTS: Stimulated endothelial-dependent relaxation of epicardial left anterior descending artery to incremental concentrations of acetylcholine in the no-ischemic preconditioning animals was shifted to the right, and maximal relaxation was attenuated compared with the nonischemic left circumflex coronary artery (117% +/- 4% vs 138% +/- 5%). In contrast, acetylcholine-induced maximal relaxation was comparable in the left anterior descending artery versus left circumflex coronary artery in the ischemic preconditioning group (130% +/- 6% vs 135% +/- 5%). In 150- to 200- microm left anterior descending microvessels, 50% relaxation occurred with a lower concentration (log[M]) of acetylcholine in ischemic preconditioning versus no-ischemic preconditioning (-8.0 +/- 0.4 vs -7.0 +/- 0.1) with no group differences in smooth muscle relaxation to sodium nitroprusside, suggesting endothelial-specific damage. Adherence of fluorescent labeled polymorphonuclear neutrophils to epicardial coronary artery endothelium, used as an index of basal (unstimulated) anti-polymorphonuclear neutrophil function, was significantly attenuated by ischemic preconditioning versus no-ischemic preconditioning (293 +/- 25 polymorphonuclear neutrophils/mm2 vs 528 +/- 29 polymorphonuclear neutrophils/mm2). CONCLUSION: In this minimally invasive direct coronary artery bypass grafting model, both agonist-stimulated and basal postischemic endothelial dysfunction were attenuated by ischemic preconditioning.     

Resistance of the failing dystrophic hamster heart to the cardioprotective effects of diltiazem and clentiazem: evidence of coronary vascular dysfunctions

Although hypothermia and cardioplegic cardiac arrest provide effective protection during cardiac surgery, ischemia of long duration, poor preoperative myocardial function, and ventricular hypertrophy may lead to heterogeneous delivery of cardioplegic solutions, incomplete protection, and impaired postischemic recovery. Calcium antagonists are potent cardioprotective agents, but their efficacy in the presence of cold cardioplegia is still controversial, especially in heart failure, since it is often believed that failing hearts are more sensitive to their negative inotropic and chronotropic actions. However, recent data have demonstrated that the benzothiazepine-like calcium antagonists diltiazem and clentiazem, in selected dose ranges, elicit significant cardioprotection independently of intrinsic cardiodepression, thus lending support to their use in cardioprotective maneuvers involving the failing heart. We therefore evaluated the cardioprotective interaction of diltiazem, clentiazem, and cold cardioplegia in both normal and failing ischemic hearts. Hearts were excised from 200- to 225-day-old cardiomyopathic hamsters (CMHs) of the UM-X7.1 line and age-matched normal healthy controls. Ex vivo perfusion was performed at a constant pressure (140 cmH2O; 1 cmH2O = 98.1 Pa) according to the method of Langendorff. Heart rate, left ventricular developed pressure (LVDP), and coronary flow were monitored throughout the study. Global ischemia was produced for 90 min by shutting down the perfusate flow, followed by reperfusion for 30 min. Normal and failing CMH hearts were either untreated (control) or perfused at the onset of global ischemia with one of the following combinations: cold cardioplegia alone (St. Thomas' Hospital cardioplegic solution, 4 degrees C, infused for 2 min), cold cardioplegia + 10 nM diltiazem, or cold cardioplegia + 10 nM clentiazem. The cardiac and coronary dilator properties of 10 nM diltiazem and 10 nM clentiazem alone were investigated in separate groups of isolated preparations. Failing CMH hearts had lower basal LVDP (42 +/- 2 vs. 77 +/- 2 mmHg (1 mmHg = 133.3 Pa) for normal hearts, p < 0.05), while coronary flow was only slightly reduced (5.6 +/- 0.2 vs. 6.2 +/- 0.2 mL/min for normal hearts). Following 90 min global ischemia, coronary flow was increased in both groups, but the peak hyperemic response declined only in failing CMH hearts (+50 +/- 17 vs. +82 +/- 17% in normal hearts). In normal hearts, LVDP virtually recovered within 5 min of reperfusion but steadily decreased thereafter (-37 +/- 4% at 30 min). In contrast, in failing CMH hearts, LVDP significantly decreased early during reperfusion but improved over time (-19 +/- 7% at 30 min). In normal hearts, the addition of diltiazem or clentiazem to cold cardioplegic solutions resulted in improved postischemic contractile function for the duration of reperfusion (85 +/- 4% vs. only 71 +/- 6% for cardioplegia, p < 0.05). The post-ischemic increase in coronary flow was similar in all groups. In failing CMH hearts, the addition of diltiazem or clentiazem afforded no significant contractile benefit at reperfusion. In nonischemic normal hearts, infusion of diltiazem or clentiazem (10 nM) alone increased coronary flow (+6 +/- 1% for diltiazem and +24 +/- 3% for clentiazem) without significant negative inotropic or chronotropic effects. In nonischemic failing CMH hearts, infusion of diltiazem or clentiazem did not elicit cardiodepression. In contrast their coronary dilator actions reverted to vasoconstriction (diltiazem) or were significantly attenuated (clentiazem). From these experiments we can conclude that, compared with the normal heart, the failing CMH heart adapted differently to global ischemia.     

Coordinate interaction between ATP-sensitive K+ channel and Na+, K+-ATPase modulates ischemic preconditioning

BACKGROUND: We reported that digoxin abolishes the infarct size (IS)-limiting effect of ischemic preconditioning (IPC). Because ATP-sensitive K+ (KATP) channels are involved in IPC, we studied whether Na+,K+-ATPase and KATP channels functionally interact, thereby modulating IPC. METHODS AND RESULTS: Rabbits received 30 minutes of coronary artery occlusion followed by 3 hours of reperfusion. IPC was elicited by 5 minutes of occlusion followed by 10 minutes of reperfusion. The IS, expressed as a percentage of the area at risk, was 40.2+/-2.8% in control and 39.8+/-5.0% in digoxin pretreatment rabbits. Both IPC and pretreatment with cromakalim, a KATP channel opener, reduced IS to 11.8+/-1.8% and 13.4+/-2.6% (P<0. 05 versus control). Digoxin abolished the reduction in IS induced by IPC (33.5+/-3.3%), whereas it did not change that induced by cromakalim (18.8+/-3.0%). In patch-clamp experiments, digoxin was found to inhibit the opening of KATP channels in single ventricular myocytes in which ATP depletion had been induced by metabolic stress. In contrast, digoxin had little effect on the channel opening induced by cromakalim. Moreover, the inhibitory action of digoxin on channel activities was dependent on subsarcolemmal ATP concentration. CONCLUSIONS: The IS-limiting effect of IPC is modulated by an interaction between KATP channels and Na+,K+-ATPase through subsarcolemmal ATP.     

The role of protein kinase in C ischemic/reperfused preconditioned isolated rat hearts

Protein kinase C (PKC) has been implicated in the preconditioning-induced cardiac protection in ischemic/reperfused myocardium. We studied the effect of PKC inhibition with calphostin C (25, 50, 100, 200, 400, and 800 nM), a potent and specific inhibitor of PKC, in isolated working nonpreconditioned and preconditioned ischemic/reperfused hearts. In the nonpreconditioned groups, all hearts underwent 30 min of normothermic global ischemia followed by 30 min of reperfusion. In the preconditioned groups, hearts were subjected to four cycles of ischemic preconditioning by using 5 min of ischemia followed by 10 min reperfusion, before the induction of 30 min ischemia and reperfusion. At low concentrations of calphostin C (25, 50, and 100 nM), the PKC inhibitor had no effect on the incidence or arrhythmias or postischemic cardiac function in the nonpreconditioned ischemic/reperfused groups. With 200 and 400 nM of calphostin C, a significant increase in postischemic function and a reduction in the incidence of arrhythmias were observed in the nonpreconditioned ischemic/reperfused groups. Increasing the concentration of calphostin C to 800 NM, the recovery of postischemic cardiac function was similar to that of the drug-free control group. In preconditioned hearts, lower concentrations (< 100 nM) of calphostin C did not change the response of the myocardium to ischemia and reperfusion in comparison to the preconditioned drug-free myocardium. Two hundred and 400 nM of the PKC inhibitor further reduced the incidence of ventricular fibrillation (VF) from the preconditioned drug-free value of 50% to 0 (p < 0.05) and 0 (p < 0.05), respectively, indicating that the combination of the two, preconditioning and calphostin C, affords significant additional protection. Increasing the concentration of calphostin C to 800 nM blocked the cardioprotective effect of preconditioning (100% incidence of VF). The recovery of cardiac function was similarly improved at calphostin C doses of 200 and 400 nM and was reduced at 800 nM (p < 0.05). With 200 and 400 nM of calphostin C, both cytosolic and particulate PKC activity were reduced by approximately 40 and 60%, respectively, in both preconditioned and preconditioned/ischemic/reperfused hearts. The highest concentration of calphostin C (800 nM) resulted in almost a complete inhibition of cytosolic (100%) and particulate (85%) PKC activity correlated with the abolition of preconditioning-induced cardiac protection. In conclusion, calphostin C protects the ischemic myocardium obtained from intact animals, provides significant additional protection to preconditioning at moderate doses, and blocks the protective effect of preconditioning at high concentrations. The dual effects of calphostin C appear to be strictly dose and "enzyme inhibition" related.     

Transient c-fos gene expression in cerebellar development and functional stimulation

The effect of ischemic preconditioning and superoxide dismutase (SOD) on endothelial glycocalyx and endothelium-dependent vasodilation in the postischemic isolated guinea-pig hearts was examined. Seven groups of hearts were used: group 1 underwent sham aerobic perfusion; group 2 was subjected to 40 min global ischemia without reperfusion; group 3, 40 min ischemia followed by 40 min reperfusion; group 4 was preconditioned with three cycles of 5 min global ischemia followed by 5 min of reperfusion (IPC), prior to 40 min ischemia; group 5 was subjected to IPC prior to standard ischemia/ reperfusion; group 6 underwent standard ischemia/reperfusion and SOD infusion (150 U/ml) was begun 5 min before 40 min ischemia and continued during the initial 5 min of the reperfusion period; group 7 was subjected to 80 min aerobic perfusion with NO-synthase inhibitor, L-NAME, to produce a model of endothelial dysfunction independent from the ischemia/reperfusion. Coronary flow responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were used as measures of endothelium-dependent and endothelium-independent vascular function, respectively. Reduction in coronary flow caused by NO-synthase inhibitor, L-NAME, served as a measure of a basal endothelium-dependent vasodilator tone. After completion of each experimental protocol, the hearts were stained with ruthenium red or lanthanum chloride for electron microscopy evaluation of the endothelial glycocalyx. While ischemia led only to a slightly flocculent appearance of the glycocalyx, in ischemia/reperfused hearts the glycocalyx was disrupted, suggesting that it is the reperfusion injury which leads to the glycocalyx injury. Moreover, the coronary flow responses to ACh and L-NAME were impaired, while the responses to SNP were unchanged in the ischemia/reperfused hearts. The disruption of the glycocalyx and the deterioration of ACh and L-NAME responses was prevented by IPC. In addition, the alterations in the glycocalyx and the impairment of ACh responses were prevented by SOD. The glycocalyx appeared to be not changed in the hearts subjected to 80 min aerobic perfusion with L-NAME. In conclusion: (1) the impairment of the endothelium-dependent coronary vasodilation is paralleled by the endothelial glycocalyx disruption in the postischemic guinea-pig hearts; (2) both these changes are prevented by SOD, suggesting the role of free radicals in the mechanism of their development; (3) both changes are prevented by IPC. We hypothesize, therefore, that alterations in the glycocalyx contribute to the mechanism of the endothelial dysfunction in the postischemic hearts.     

Cerebellar granule cell GABA(A) receptors studied at the single-channel level: modulation by protein kinase G

BACKGROUND: This study was designed to evaluate the adenosine-triphosphate-sensitive potassium channel opener pinacidil as a blood cardioplegic agent. METHODS: Using a blood-perfused, parabiotic, Langendorff rabbit model, hearts underwent 30 minutes of normothermic ischemia protected with blood cardioplegia (St. Thomas' solution [n = 8] or Krebs-Henseleit solution with pinacidil [50 micromol/L, n = 81) and 30 minutes of reperfusion. Percent recovery of developed pressure, mechanical arrest, electrical arrest, reperfusion ventricular fibrillation, percent tissue water, and myocardial oxygen consumption were compared. RESULTS: The percent recovery of developed pressure was not different between the groups (52.3 +/- 5.9 and 52.8 +/- 6.9 for hyperkalemic and pinacidil cardioplegia, respectively). Pinacidil cardioplegia was associated with prolonged electrical and mechanical activity (14.4 +/- 8.7 and 6.1 +/- 3.9 minutes), compared with hyperkalemic cardioplegia (1.1 +/- 0.6 and 1.1 +/- 0.6 minutes, respectively; p < 0.05). Pinacidil cardioplegia was associated with a higher reperfusion myocardial oxygen consumption (0.6 +/- 0.1 versus 0.2 +/- 0.0 mL/100 g myocardium/beat; p < 0.05) and a higher percent of tissue water (79.6% +/- 0.7% versus 78.6% +/- 1.2%; p < 0.05). CONCLUSIONS: Systolic recovery was not different between groups, demonstrating comparable effectiveness of pinacidil and hyperkalemic warm blood cardioplegia.     

Preconditioning during simulated MIDCABG attenuates blood flow defects and neutrophil accumulation

BACKGROUND: Ischemic preconditioning (IP) may be cardioprotective in minimally invasive direct coronary artery bypass where cardioplegia is not used. This study tested the hypothesis that IP of the area at risk (AAR) would attenuate postischemic injury from transient coronary artery occlusion. METHODS: In 19 anesthetized dogs, the left anterior descending coronary artery was occluded for 30 minutes (simulating coronary occlusion during anastomosis) followed by 3 hours of reperfusion. In 10 dogs, occlusion was preceded by 5 minutes of occlusion and 5 minutes of reperfusion (IP), whereas 9 other dogs had no IP (control, C). RESULTS: Thirty minutes of left anterior descending occlusion caused comparable dyskinesis (systolic shortening, sonomicrometry) in the AAR in C (baseline, 29% +/- 3% to 3% +/- 2%) and in IP (baseline, 29% +/- 2% to -0.3% +/- 2%). After 3 hours of reperfusion, systolic shortening was significantly depressed in C (20% +/- 4%), and was not significantly improved by IP (24% +/- 3%, p = 0.8 versus C). Postischemic diastolic stiffness in the AAR was not altered by IP versus C (0.60 +/- 0.12 versus 0.41 +/- 0.13). Plasma creatine kinase activity was similar between C and IP at the end of reperfusion (20 +/- 11 versus 16 +/- 5 U/g). Postischemic AAR blood flow (in milliliters per minute per gram of tissue) at 180 minutes of reperfusion decreased by 56% versus baseline in C (from 1.04 +/- 0.4 to 0.46 +/- 0.12; p < 0.05) compared with no change in IP (from 0.74 +/- 0.23 to 0.60 +/- 0.10), but there was no significant group difference at this time. Myeloperoxidase activity as an index of neutrophil accumulation in AAR was decreased in IP versus C (0.4 +/- 0.09 versus 0.7 +/- 0.04 U/microg tissue). CONCLUSIONS: Ischemic preconditioning does not decrease postischemic wall motion and only modestly increases postischemic blood flow abnormalities in the AAR, but does significantly inhibit neutrophil accumulation.     

Coronary flow reserve after ischemia and reperfusion of the isolated heart. Divergent results with crystalloid versus blood perfusion

Mechanical function and coronary hemodynamics were assessed in 73 isolated rabbit hearts randomly subjected to 0, 10, 20, 30, or 45 minutes of 37 degrees C global ischemia and 45 minutes of reperfusion in either a modified Krebs buffer or homologous blood-perfused Langendorff mode (n = 7 to 9 hearts per group). Isovolumic developed pressure, resting coronary flow, and response to endothelium-dependent (bradykinin) and -independent (nitroglycerin) agonists were quantitated at defined preload and heart rate. Perfusate did not influence systolic performance, which was impaired after 30 minutes of ischemia and fell to 64% to 72% of preischemic values after 45 minutes of ischemia (p < 0.05). However, basal coronary flow was at least sixfold greater in crystalloid-perfused hearts. Moreover, coronary hyperemia (p < 0.05) persisted for Krebs-perfused hearts subjected to all but the longest ischemic interval. After equilibration, all postischemic blood-perfused hearts had basal flow unchanged from before ischemia. Bradykinin and nitroglycerin induced similar increases in coronary flow for each group before and after each ischemia interval. However, the magnitude of this increase was greater in blood-perfused hearts (p < 0.01) and was not attenuated by the ischemic times encompassed in this protocol. In contrast, endothelium-dependent and -independent coronary flow reserve was abolished after 20 minutes of ischemia or longer in Krebs-perfused hearts. These data suggest that the unphysiologic resting flow patterns of crystalloid-perfused isolated hearts obfuscate interpretation of the interaction between coronary flow reserve and ischemic injury.     

Calcium preconditioning in human myocardium

BACKGROUND: Ischemic stress and other protein kinase C (PKC)-linked receptor stimuli can induce rapid cardiac protection against ischemia-reperfusion injury. We and others have demonstrated that exogenous calcium (Ca2+) pretreatment confers PKC-mediated cardiac functional and infarct protection in animal models, but it remains unknown whether Ca2+ preconditioning confers similar postischemic functional protection in human myocardium, and, if so, whether the mechanism is mediated by PKC. We postulated that Ca2+ preconditioning confers ischemic tolerance to human myocardium by a PKC-dependent mechanism. METHODS: Human atrial trabeculae were suspended in organ baths and paced at 1 Hz, and force development was recorded. After 90 minutes of equilibration, all trabeculae were subjected to ischemia (45 minutes) and reperfusion (120 minutes). Exogenous CaCl2 (3.0 mmol/L for 5 minutes) or vehicle (saline solution) was administered before simulated ischemia, with or without concurrent PKC inhibition (bisindolylmaleimide I, 150 nmol/L). RESULTS: Ischemia-reperfusion resulted in decreased postischemic developed force, Ca2+ preconditioning protected human myocardium against ischemia-reperfusion injury (p < 0.05 versus control ischemia-reperfusion), and concurrent PKC inhibition abolished the salutary effect of Ca2+ preconditioning in human myocardium (p < 0.05 versus Ca2+ preconditioning). CONCLUSIONS: Preconditioning with Ca2+ represents a potent means of accessing PKC-mediated protection of the human myocardium against ischemia-reperfusion injury.     

Delayed effects of sublethal ischemia on the acquisition of tolerance to ischemia

The infarct-limiting effect of ischemic preconditioning is believed to be a transient phenomenon. We examined the delayed effects of repetitive brief ischemia on limiting infarct size in an open-chest dog model by an occlusion (90 minutes) of the left anterior descending coronary artery (LAD) followed by reperfusion (5 hours). The dogs were preconditioned with four brief repeated ischemic episodes induced by 5-minute LAD occlusions with subsequent reperfusion. The size of infarcts initiated by a sustained occlusion immediately or 24 hours after preconditioning was significantly smaller when compared with infarcts in sham-operated dogs (for the immediate occlusion, 14.4 +/- 2.0% versus 39.0 +/- 3.7%, respectively [p < 0.01]; and for the delayed occlusion, 18.8 +/- 3.4% versus 35.1 +/- 4.6%, respectively [p < 0.05]); however, when the infarction was induced 3 hours (31.2 +/- 3.7% versus 37.5 +/- 4.2%, respectively) or 12 hours (25.4 +/- 4.8% versus 35.0 +/- 5.3%, respectively) after repetitive ischemia, the infarct size did not differ. No differences were seen in regional myocardial blood flow or rate-pressure products between the two groups. These results indicate that an infarct-limiting effect of brief repeated ischemia can be observed 24 hours after sublethal preconditioning.     

Importance of endogenous adenosine during ischemia and reperfusion in neonatal porcine hearts

BACKGROUND: Adenosine has several potentially cardioprotective effects. We hypothesized that the effects of endogenous adenosine vary with degree of ischemia and that elevating endogenous levels is protective. METHODS AND RESULTS: Isolated blood-perfused piglet hearts underwent 120 minutes of low-flow ischemia (10% flow) or 90 minutes of zero-flow ischemia, all with 60 minutes of reperfusion. Hearts were treated with either saline, the adenosine receptor blocker 8-sulfophenyltheophylline (8SPT, 300 micromol x L(-1)), or the nucleoside transport inhibitor draflazine (1 micromol x L(-1)). In separate groups, biopsies were obtained before and at the end of ischemia. Compared with saline, 8SPT did not significantly alter functional recovery in either protocol. Draflazine significantly improved percent recovery of left ventricular systolic pressure both in the low-flow protocol (92+/-3% versus 75+/-2% [saline] and 73+/-3% [8SPT], P<.001 for both) and in the zero-flow protocol (76+/-3% versus 59+/-4% [saline] and 46+/-9% [8SPT], P<.05 for both). In the zero-flow protocol, draflazine also significantly reduced ischemic contracture and release of creatine kinase. Tissue adenosine at the end of ischemia was elevated by draflazine compared with saline-treated hearts: after low-flow ischemia to 0.10+/-0.05 versus 0.00+/-0.00 micromol x g(-1) dry wt (P<.05) and after zero-flow ischemia to 1.73+/-0.82 versus 0.15+/-0.03 micromol x g(-1) dry wt (P<.05). CONCLUSIONS: In neonatal porcine hearts, endogenous adenosine produced during ischemia does not influence ischemic injury or functional recovery. Elevating endogenous adenosine by draflazine elicits cardioprotection in both low-flow and zero-flow conditions.     

New strategies for intraoperative myocardial protection

In the past, most strategies for intraoperative myocardial protection were developed in models using nondiseased adult hearts from various animal species. In the clinical setting, however, myocardial status in cardiac patients may be quite different and there is a need to adapt our current protective strategies to the actual pathophysiological status of the heart. In the immature heart as well as in the senescent heart, current protective techniques have been shown to be deficient and further research is required. New insights have been gained into the pathophysiological processes underlying chronic ischemic left ventricular dysfunction in the "hibernating" myocardium. It has been shown that viability in these hearts is associated with subcellular alterations related to dedifferentiation of the myocytes. This finding explains the delayed recovery in function of these hearts after revascularization and the need for intraoperative protective strategies focusing on the prevention of stunning in the nonhibernating segments. Tepid continuous retrograde blood cardioplegia is suggested as the optimal technique. Unraveling the mechanisms of preconditioning in the heart and understanding endogenous myocardial protection may provide clues for novel cardioprotective techniques. Adenosine itself may be used as an adjunct to cardioplegia, and treatment with adenosine regulating agents or nucleoside transport inhibitors shows promising results. Like adenosine, other hyperpolarizing agents (potassium-channel openers) are suggested for arrest of the heart instead of the depolarizing agents commonly used in cardioplegia. Finally, the role of Na(+)-H+ exchange in the development of ischemic and postischemic injury has become more clear. By the use of a new selective Na(+)-H+ exchange inhibitor, postischemic contracture can be dramatically reduced and contractility improved. This opens prospective approaches in emergency coronary bypass surgery for evolving myocardial infarction.     

Quality assurance in heart surgery. General and personal concepts

Prolonged tissue ischemia and subsequent reperfusion result in significant tissue injury due to the ischemic-reperfusion syndrome. Although skeletal muscle has significant tolerance to ischemic-reperfusion injury (IRI), compared to other organ systems, IRI of skeletal muscle does occur when there is a prolonged ischemic period. In many reconstructive surgical procedures involving microsurgery and prolonged tissue ischemia time, IRI-induced skeletal-muscle injury is a serious clinical concern. Specifically, there are significant vascular complications (venous thrombosis and arteriolar no-reflow) and loss of transplanted muscle function on reperfusion with prolonged ischemia. Ischemic preconditioning (IPC) or adenosine (ADO) pretreatment applied prior to the ischemic period are known to protect against IRI in cardiac muscle. Recent data from basic science research suggest that IPC or ADO pretreatment may be employed to protect skeletal muscle against IRI. This review summarizes the basic mechanisms and potential clinical relevance of ischemia- and reperfusion-induced skeletal muscle injury and describes how skeletal muscle can be protected against IRI with IPC or ADO pretreatment.     

Changes in ischemic tolerance and effects of ischemic preconditioning in middle-aged rat hearts

BACKGROUND: Although both clinical and animal studies have shown that ischemic tolerance is reduced in the senescent myocardium, it has not been clarified when myocardium becomes more vulnerable to ischemia. Preconditioning protects the hearts of young adult animals of various species, but its effects are not identical in human studies. We investigated whether ischemic tolerance and the effect of preconditioning decreased in isolated hearts of middle-aged rats. METHODS AND RESULTS: The hearts of young adult rats (12 weeks old: group Y, n = 44) and middle-aged rats (50 weeks old: group M, n = 44) were subjected to global ischemia for 15, 20, or 25 minutes followed by reperfusion. Hearts were also subjected to preconditioning and then to 20 (group Y, n = 22) or 15 (group M, n = 22) minutes of ischemia followed by reperfusion. Left ventricular developed pressure (LVDP) was decreased by 40% to 60%, and the level of ATP was decreased by 60% to 70% in group M compared with group Y. Preconditioning increased LVDP (% LVDP, 40.5% to 72.4%) and levels of high-energy phosphates (ATP, 11.8 to 14.1; creatine phosphate, 17.0 to 23.1 mumol/g dry wt) and reduced left ventricular end-diastolic pressure (LVEDP, 32.8 to 10.3 mm Hg), creatine kinase release (257 to 132 U/g dry wt), and ryanodine-sensitive sarcoplasmic reticulum Ca2+ release after ischemia in group Y. Preconditioning exerted opposite effects in group M (% LVDP, 45.9% to 15.8%; LVEDP, 21.0 to 28.5 mm Hg; ATP, 14.1 to 8.5 mumol/g dry wt; and CK release, 176 to 332 U/g dry wt). Preconditioning was associated with increases in the incidence of reperfusion-induced ventricular fibrillation (0% to 62.5%) and the rate of sarcoplasmic reticulum Ca2+ release in group M. CONCLUSIONS: These results indicate that hearts became more vulnerable to ischemia with age and that the beneficial effects of preconditioning were reversed in middle-aged rat hearts.