Energy metabolism after ischemic preconditioning in streptozotocin-induced diabetic rat hearts

OBJECTIVES: The aim of this study was to compare the cardioprotective effects of preconditioning in hearts from streptozotocin-induced diabetic rats with its effects in normal rat hearts. BACKGROUND: The protective effect of ischemic preconditioning against myocardial ischemia may come from improved energy balance. However, it is not known whether preconditioning can also afford protection to diabetic hearts. METHODS: Isolated perfused rat hearts were either subjected (preconditioned group) or not subjected (control group) to preconditioning before 30 min of sustained ischemia and 30 min of reperfusion. Preconditioning was achieved with two cycles of 5 min of ischemia followed by 5 min of reperfusion. RESULTS: In the preconditioned groups of both normal and diabetic rats, left ventricular developed pressure, high energy phosphates, mitochondrial adenosine triphosphatase and adenine nucleotide translocase activities were significantly preserved after ischemia-reperfusion; cumulative creatine kinase release was smaller during reperfusion; and myocardial lactate content was significantly lower after sustained ischemia. However, cumulative creatine kinase release was less in the preconditioned group of diabetic rats than in the preconditioned group of normal rats. Under ischemic conditions, more glycolytic metabolites were produced in the diabetic rats (control group) than in the normal rats, and preconditioning inhibited these metabolic changes to a similar extent in both groups. CONCLUSIONS: The present study demonstrates that in both normal and diabetic rats, preservation of mitochondrial oxidative phosphorylation and inhibition of glycolysis during ischemia can contribute to preconditioning-induced cardioprotection. Furthermore, our data suggest that diabetic myocardium may benefit more from preconditioning than normal myocardium, possibly as a result of the reduced production of glycolytic metabolites during sustained ischemia and the concomitant attenuation of intracellular acidosis.     

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 preconditional protection of ischemia reperfusion injury on isolated hearts of ground squirrel and rat

The protective effects of ischemic preconditioning on ischemia-reperfusion injury was investigated using isolated Langendorff perfusing hearts from ground squirrel and rat. In Preconditioning I group hearts were first perfused with Krebs-Henseleit solution for 10 min to establish a steady state, then stopped for 15 min to establish global ischemia, and finally followed by 10 min ischemia and 10 min reperfusion. In Preconditioning II group there were three cycles of 5 min ischemia + 5 min reperfusion after 10 min equilibration and then the final 10 min ischemia and 10 min reperfusion were followed. It was found that in group I during the final 10 min ischemia period there was remarkable augmentation of CK release from both animal's hearts. But in group II CK release decreased markedly during the same ischemic period. CK release during final 10 min reperfusion period also decreased significantly in group II in comparison with group I. The incidence of arrhythmias occurred in both animal's hearts was markedly reduced in group II rather than group I. In conclusion, short episode ischemic preconditioning protect subsequent ischemia-reperfusion injury on isolated hearts from ground squirrel and rat.     

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.     

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.     

Transgenic A1 adenosine receptor overexpression markedly improves myocardial energy state during ischemia-reperfusion

A1 adenosine (A1AR) activation may reduce ischemia-reperfusion injury. Metabolic and functional responses to 30 min global normothermic ischemia and 20 min reperfusion were compared in wild-type and transgenic mouse hearts with approximately 100-fold overexpression of coupled cardiac A1ARs. 31P-NMR spectroscopy revealed that ATP was better preserved in transgenic v wild-type hearts: 53 +/- 11% of preischemic ATP remained after ischemia in transgenic hearts v only 4 +/- 4% in wild-type hearts. However, recovery of ATP after reperfusion was similar in transgenic (46 +/- 5%) and wild-type hearts (37 +/- 12%). Reductions in phosphocreatine (PCr) and cytosolic pH during ischemia were similar in both groups. However, recovery of PCR on reperfusion was higher in transgenic (67 +/- 8%) v wild-type hearts (36 +/- 8%), and recovery of pH was greater in transgenic (pH = 7.11 +/- 0.05) v wild-type hearts (pH = 6.90 +/- 0.02). Bioenergetic state ([ATP]/[ADP].[Pi]) was higher in transgenic v wild-type hearts during ischemia-reperfusion. Time to ischemic contracture was prolonged in transgenic (13.6 +/- 0.8 min) v wild-type hearts (10.4 +/- 0.3 min). Degree of contracture was lower and recovery of function in reperfusion higher in transgenic v wild-type hearts. In conclusion, A1AR overexpression reduces ATP loss and improves bioenergetic state during severe ischemic insult and reperfusion. These changes may contribute to improved functional tolerance.     

Aldose reductase inhibition protects diabetic and nondiabetic rat hearts from ischemic injury

Diabetes increases the incidence of cardiovascular disease as well as the complications of myocardial infarction. Studies using animal models of diabetes have demonstrated that the metabolic alterations occurring at the myocyte level may contribute to the severity of ischemic injury in diabetic hearts. Of the several mechanisms being investigated to understand the pathogenesis of diabetic complications, the increased metabolism of glucose via the polyol pathway has received considerable attention. Deviant metabolic regulation due to increased flux through aldose reductase in diabetic hearts may influence the ability of the myocardium to withstand ischemia insult. To determine if aldose reductase inhibition improves tolerance to ischemia, hearts from acute type I diabetic and nondiabetic control rats were isolated and retrograde perfused. Each group was exposed to 1 micromol/l zopolrestat, a specific inhibitor of aldose reductase, for 10 min, followed by 20 min of global ischemia and 60 min of reperfusion in the absence of zopolrestat. Zopolrestat reduced sorbitol levels before ischemia in diabetic hearts. The cytosolic redox state (NADH/NAD+), as measured by lactate-to-pyruvate ratios, was significantly lowered under baseline, ischemic, and reperfusion conditions in diabetic hearts perfused with zopolrestat. In these diabetic hearts, ATP was significantly higher in zopolrestat hearts during ischemia, as were phosphocreatine and left ventricular-developed pressure on reperfusion. Zopolrestat provided similar metabolic and functional benefits in nondiabetic hearts. Creatine kinase release was reduced by approximately 50% in both nondiabetic and diabetic hearts treated with zopolrestat. These data indicate that inhibition of aldose reductase activity preserves high-energy phosphates, maintains a lower cytosolic NADH/NAD+ ratio, and markedly protects both diabetic and nondiabetic hearts during ischemia and reperfusion.     

Low concentrations of adenosine receptor blocker decrease protection by hypoxic preconditioning in ischemic rat hearts

A role for adenosine in ischemic preconditioning and hypoxic preconditioning (HP) has been established in several species but is controversial in rats, due in part to the inconsistency of the data from the different experimental design. Our objective was to investigate the role of adenosine in the protection of the ischemic myocardium by HP in rats. Methods: perfused hearts isolated from Sprague-Dawley rats were exposed to 5 min of hypoxic perfusion before 25 min of global ischemia followed by 20 min of reperfusion. The effects of adenosine receptor antagonist, 8-(p-sulfophenyl)-theophylline (8SPT) on HP-based changes in left-ventricular function, energy metabolites, and release of creatine kinase and lactate dehydrogenase were determined. To minimise non-specific effects of 8SPT, low concentrations of agent (0.5 or 1.0 micro mol/l) were used. Results: 8SPT alone had no deleterious effects on normoxically perfused hearts or on ischemic/reperfused hearts. HP improved the recovery of LV function and creatine phosphate, and reduced the release of enzymes during reperfusion. 8SPT (1.0 micromol/l) ameliorated the beneficial effect of HP on cardiac function, but did not reverse the reduction in release of enzymes by HP completely. Conclusion: results suggest that the protective effect of HP on myocardial contractile function may be mediated by receptor(s) that can be inhibited by low concentrations of antagonist but may not have a primary role in the reduction of cellular damage by HP in rats.     

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.     

Aging impairs functional, metabolic and ionic recovery from ischemia-reperfusion and hypoxia-reoxygenation

Functional and metabolic responses to ischemia-reperfusion and hypoxia-reoxygenation were studied in Langendorff perfused hearts from mature (2-4 months) and aged (18-24 months) Wistar rats. Hearts were subjected to 20 min global ischemia or hypoxia followed by 30 min reperfusion or reoxygenation. Cellular metabolism was assessed by 31P-NMR spectroscopy. Normoxic function, phosphate metabolite levels, and cytosolic free energy state (delta GATP) were comparable in both age groups, although free [5'-AMP] and purine efflux were elevated in aged hearts. There were no aging-related differences in phosphate metabolite levels, pH or delta GATP during ischemia or hypoxia. Nevertheless, ischemic and hypoxic contracture tended to be higher in aged hearts. After reperfusion, heart rate x left-ventricular pressure recovered to 55% of pre-ischemia in mature hearts, and only 25% in aged hearts. After reoxygenation, function recovered to 75% in mature hearts and 55% in aged hearts. Recoveries of cellular [ATP], [phosphocreatine], [inorganic phosphate] and [Mg2+] were impaired, and delta GATP was consistently depressed in aged v mature hearts, Impaired recovery of delta GATP was associated with enhanced purine efflux in aged hearts. Post-ischemic Na+ and Ca2+ accumulation was also increased by 30-40% in aged hearts. Tissue damage assessed by post-ischemic creatine kinase efflux was modest in mature hearts (< 2% total tissue activity) and was 2.5-fold higher in aged hearts. The data show that: (i) aging reduces contractile recovery from ischemia/hypoxia; (ii) this is unrelated to the metabolic insult during ischemia/hypoxia, but parallels reduced recovery of delta GATP [inorganic phosphate], [Mg2+]i [Na+] and [Ca2+]; and (iii) increased purine catabolism may contribute to poor metabolic recovery in aged hearts.     

Activation of cardiac muscarinic receptor and ischemic preconditioning effects in in situ rat heart

Activation of cardiac muscarinic receptors by vagal stimulation decreases cardiac work, which may have a protective effect against ischemic injury. To determine whether cardiac muscarinic receptors contribute to the mechanisms of preconditioning effects, we examined the effect of carbachol on ischemia/reperfusion damage and the effect of vagotomy on cardioprotection induced by ischemic preconditioning. Rats were subjected to 30 min of left coronary artery occlusion followed by 30-min reperfusion in situ. Pre-conditioning was induced by three cycles of 2-min coronary artery occlusion and, subsequently by 5 min of reperfusion. The incidence of ischemic arrhythmias, such as ventricular tachycardia (VT) and ventricular fibrillation (VF), and the development of myocardial infarction were markedly reduced by the preconditioning. Carbachol infusion (4 micrograms/kg per min) delayed the occurrence of VT and VF during ischemia and reduced the infarct size. Compared with non-ischemic left ventricle, the cyclic guanosine monophosphate (GMP) content in the ischemic region of the left ventricle was decreased by ischemia/reperfusion, whereas the cyclic adenosine monophosphate (AMP) content of this region was increased. These changes were reversed by preconditioning. Similar changes in cyclic GMP and AMP content in the ischemic region were seen in rats undergoing carbachol treatment. These results suggest the possible contribution of muscarinic receptor stimulation to preconditioning. Vagotomy prior to preconditioning diminished the antiarrhythmic effects, whereas it did not block the anti-infarct effect afforded by pre-conditioning. Vagotomy abolished the preconditioning effect on the tissue cyclic GMP, but it did not attenuate the decrease in tissue cyclic AMP. The results suggest that muscarinic stimulation exerts preconditioning-mimetic protective effects in ischemic/reperfused hearts, but that a contribution of reflective vagal activity to the mechanism for preconditioning is unlikely.     

Role of increased cytosolic free calcium concentration in myocardial ischemic injury

Increases in cytosolic free calcium concentration ([Ca2+]I) may play an important role in myocardial ischemic injury. An early effect of the rise in [Ca2+]I may be impaired postischemic contractile function if the ischemic myocardium is reperfused during the reversible phase of ischemic injury; furthermore, if the rise in [Ca2+]I is prolonged, a cascade of events may be initiated which ultimately results in lethal injury. With the development of methods for measuring [Ca2+]I, it has become possible to evaluate directly the role of increased [Ca2+]I in myocardial ischemic injury. Although it has been possible to show that inhibition of the transport processes which contribute to the early rise in [Ca2+]I attenuates stunning and the rise in [Ca2+]I concurrently, if increased [Ca2+]I plays an important role in ischemic injury, then it should be possible to show that interventions which alter the timecourse of ischemic injury also alter the timecourse of the rise in [Ca2+]I in a parallel manner. Recently, considerable effort has been expended to investigate the mechanisms underlying the preconditioning phenomenon, whereby repetitive brief periods of ischemia prior to a sustained period of ischemia protects the myocardium from injury during the sustained period of ischemia, and this has stimulated additional work to understand the possible involvement of adenosine as a mediator of preconditioning as well as to understand the protective effects of adenosine. Measurements of [Ca2+]I using 19F NMR of 5FBAPTA-loaded hearts have shown that preconditioning attenuates the rise in [Ca2+]I during 30 min of ischemia and reduces stunning during reflow. Adenosine pretreatment mimics the effects of preconditioning on the rise in [Ca2+]I and on stunning, but adenosine receptor antagonists do not eliminate the protective effects of preconditioning, although some adenosine antagonists also block hexose transport and under these conditions, the ability of preconditioning to attenuate the rise in [Ca2+]I is abolished and there is a corresponding loss of the protective effect of preconditioning on stunning. Although it has been suggested that the beneficial effect of preconditioning on infarct size can be eliminated by pretreatment with glibenclamide, in the isolated rat heart glibenclamide does not affect the attenuation of the rise in [Ca2+]I induced by preconditioning and does not affect stunning. All of these studies show a consistent relationship between the magnitude of the rise in [Ca2+]I during ischemia and the degree of stunning during reperfusion. The data suggest that increased [Ca2+]I plays a very important role in myocardial ischemic injury.     

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.     

Contrast enhanced sonography of visceral perfusion defects in dogs

The role of adrenergic mechanism in the cardioprotective effect of ischemic preconditioning against ischemia-reperfusion induced injury in in vivo dog heart and isolated rat heart was investigated. Anesthetized dogs were subjected to LAD coronary artery ligation for 60 min followed by reperfusion for 4 h. Preconditioning protocol was 5 min of ischemia followed by reperfusion for 10 min. Rat hearts were subjected to global ischemia for 30 min followed by reperfusion for 30 min. Preconditioning protocol was 5 min global ischemia followed by reperfusion for 5 min repeated four times. Infarct size, electrocardiographic changes and release of LDH were estimated to assess the extent of cardiac injury. Preconditioning reduced the infarct size, ST segment elevation and prevented the loss of R wave. Prazosin attenuated the cardioprotective effect of preconditioning in dog. Preconditioning conferred protection against ischemia-reperfusion induced cardiac injury and reperfusion-induced arrhythmias in isolated rat heart. Reserpine pretreatment attenuated this protective effect of preconditioning on reperfusion-induced arrhythmias. These observations suggest the involvement of adrenergic mechanism in the cardioprotective and antiarrhythmic effect of ischemic preconditioning in dog and rat species respectively.     

Adenosine-enhanced ischemic preconditioning provides enhanced cardioprotection in the aged heart

BACKGROUND: Recently we have reported a novel myo-protective protocol "adenosine-enhanced ischemic preconditioning" (APC), which extends and amends the protection afforded by ischemic preconditioning (IPC) by both reducing myocardial infarct size and enhancing postischemic functional recovery in the mature rabbit heart. However, the efficacy of APC in the senescent myocardium was unknown. METHODS: The efficacy of APC was investigated in senescent rabbit hearts and compared with magnesium-supplemented potassium cardioplegia (K/Mg) and IPC. Global ischemia (GI) hearts were subjected to 30 minutes of global ischemia and 120 minutes of reperfusion. Ischemic preconditioning hearts received 5 minutes of global ischemia and 5 minutes of reperfusion before global ischemia. Magnesium-supplemented potassium cardioplegia hearts received cardioplegia just before global ischemia. Adenosine-enhanced ischemic preconditioning hearts received a bolus injection of adenosine in concert with IPC. To separate the effects of adenosine from that of APC, a control group (ADO) received a bolus injection of adenosine 10 minutes before global ischemia. RESULTS: Infarct size was significantly decreased to 18.9%+/-2.7% with IPC (p<0.05 versus GI); 17.0%+/-1.0% with ADO (p<0.05 versus GI); 7.7%+/-1.3% with K/Mg (p<0.05 versus GI, IPC, and ADO); and 2.1%+/-0.6% with APC (p<0.05 versus GI, IPC, ADO, and K/Mg; not significant versus control). Only APC and K/Mg significantly enhanced postischemic functional recovery (not significant versus control). CONCLUSIONS: Adenosine-enhanced ischemic preconditioning provides similar protection to K/Mg cardioplegia, significantly enhancing postischemic functional recovery and decreasing infarct size in the senescent myocardium.     

The multifarious spectrum of ischemic left ventricular dysfunction: relevance of new ischemic syndromes

Ischemic heart disease, once limited to a number of well defined entities such as angina of effort, unstable angina, and myocardial infarction, must now be regarded as a much more complex and elusive entity. Silent ischemia was the first of the new ischemic syndromes to be described. Recently, three further new syndromes have been added, namely stunning, hibernation and preconditioning. All three have one common theme--they can be related to ischemia and reperfusion. In stunning, there is post-reperfusion mechanical dysfunction that recovers. In hibernation, there is prominent contractile dysfunction, apparently out of proportion to the reduction in coronary flow, and the recovery upon reperfusion is good. In preconditioning, severe ischemia followed by reperfusion protects against subsequent ischemia which may modify the severity of ischemic damage in the other ischemic syndromes. Ischemic LV dysfunction as found in post-infarct patients and in the absence of any simple relation to reperfusion, can be either diastolic or systolic or both in nature. In ischemic LV diastolic dysfunction without major systolic dysfunction, calcium antagonists may be appropriate therapy which could point to a role for abnormalities in the regulation of cytosolic calcium. It is proposed that there is potentially a mixed post-infarct syndrome, which may comprise one or more of the new ischemic syndromes (silent ischemia, stunning, hibernation, and preconditioning), as well as a varying degree of systolic and/or diastolic dysfunction. The basis of the systolic dysfunction is, at least in part, post-infarct LV remodeling. Several of these entities could overlap in the same patient. The term "mixed post-infarct ischemic syndrome" is suggested to describe this condition.     

Endothelin-1 increases susceptibility of isolated rat hearts to ischemia/reperfusion injury by reducing coronary flow

Endothelin-1 (ET-1) is the most potent vasoconstrictor known to date, and it was proposed that this peptide plays a major role in myocardial ischemia/reperfusion injury. ET-1 could increase myocardial susceptibility to ischemia by two mechanisms: via coronary flow reduction and/or via direct, metabolic effects on the heart. In isolated, buffer-perfused rat hearts, function was measured with a left ventricular balloon, and energy metabolism (ATP, phosphocreatine, inorganic phosphate, intracellular pH) was estimated by 31NMR-spectroscopy. Under constant pressure perfusion, hearts were subjected to 15 min of control perfusion, 15 ("moderate injury") or 30 ("severe injury") min of global ischemia, followed by 30 min of reperfusion. Hearts were pre-treated with ET-1 (boluses of 0.04, 4, 40 of 400 pmol) 5 min prior to ischemia. In the control period, ET-1 reduced coronary flow, ventricular function, phosphocreatine and intracellular pH dose-dependently: during ischemia/reperfusion, coronary flow, functional recovery and high-energy phosphate metabolism were adversely affected by ET-1 in a dose-related manner. To study effects of ET-1 not related to coronary flow reduction, additional hearts were perfused under constant flow conditions (ET-1 0 or 400 pmol) during 15 min of control, 15 min of ischemia and 30 min of reperfusion. When coronary flow was held constant, functional and energetic parameters were similar for untreated and ET-1 treated hearts during the entire protocol, i.e. the adverse effects of ET-1 on function and energy metabolism during ischemia/reperfusion were completely abolished. In both constant pressure and constant flow protocols, 400 pmol ET-1 reduced the extent of ischemic intracellular acidosis. The authors conclude that ET-1 increases the susceptibility of isolated hearts to ischemia/reperfusion injury via reduction of coronary flow.     

Recombinant adeno-associated virus vector: use for transgene expression and anterograde tract tracing in the CNS

Several signal transduction pathways have been implicated in the mechanism of protection induced by ischemic preconditioning (PC). For example, stimulation of a variety of G-protein coupled receptors results in stimulation of protein kinase C (PKC) which has been suggested to act as common denominator in eliciting protection. PC also significantly attenuated cAMP accumulation during sustained ischemia, suggesting involvement of an anti-adrenergic mechanism. The aim of this study was to evaluate the beta-adrenergic signal transduction pathway (as evidenced by changes in tissue cAMP and cAMP- and cGMP-phosphodiesterase) during the PC protocol as well as during sustained ischemia. Isolated perfused rat hearts were preconditioned by 3 x 5 min global ischemia (PC1,2,3) interspersed by 5 min reperfusion, followed by 25 min global ischemia. Tissue cAMP- and cGMP-PDE activity as well as cAMP and cGMP levels were determined at different time intervals during the PC protocol and sustained ischemia. Tissue cAMP increased with each PC ischemic event and normalized upon reperfusion, while PDE activity showed the opposite, viz a reduction during ischemia and an increase during reperfusion. Except for PC1, tissue cGMP showed similar fluctuations. Throughout 25 min sustained ischemia, cAMP- and cGMP-PDE activities were higher in PC than in nonpreconditioned hearts, associated with a significantly lesser accumulation in cAMP and higher cGMP levels in the former. Fluctuations in cyclic nucleotides during preconditioning were associated with concomitant changes in PDE activity, while the attenuated beta-adrenergic response of preconditioned hearts during sustained ischemia may partially be due to increased PDE activity.