Transgenic A1 adenosine receptor overexpression increases myocardial resistance to ischemia

Activation of myocardial A1 adenosine receptors (A1AR) protects the heart from ischemic injury. In this study transgenic mice were created using the cardiac-specific alpha-myosin heavy chain promoter and rat A1AR cDNA. Heart membranes from two transgene positive lines displayed approximately 1,000-fold overexpression of A1AR (6,574 +/- 965 and 10,691 +/- 1,002 fmol per mg of protein vs. 8 +/- 5 fmol per mg of protein in control hearts). Compared with control hearts, transgenic Langendorff-perfused hearts had a significantly lower intrinsic heart rate (248 beats per min vs. 318 beats per min, P < 0. 05), lower developed tension (1.2 g vs. 1.6 g, P < 0.05), and similar coronary resistance. The difference in developed tension was eliminated by pacing. Injury of control hearts during global ischemia, indexed by time-to-ischemic contracture, was accelerated by blocking adenosine receptors with 50 microM 8-(p-sulfophenyl) theophylline but was unaffected by addition of 20 nM N6-cyclopentyladenosine, an A1AR agonist. Thus A1ARs in ischemic myocardium are presumably saturated by endogenous adenosine. Overexpressing myocardial A1ARs increased time-to-ischemic contracture and improved functional recovery during reperfusion. The data indicate that A1AR activation by endogenous adenosine affords protection during ischemia, but that the response is limited by A1AR number in murine myocardium. Overexpression of A1AR affords additional protection. These data support the concept that genetic manipulation of A1AR expression may improve myocardial tolerance to ischemia.     

Is a high glycogen content beneficial or detrimental to the ischemic rat heart? A controversy resolved

A high glycogen level may be beneficial to the ischemic heart by providing glycolytic ATP or detrimental by increasing intracellular lactate and protons. To determine the effect of high glycogen on the ischemic myocardium, the glycogen content of Langendorff-perfused rat hearts was either depleted or elevated before 32 minutes of low-flow (0.5 mL/min) ischemia with Krebs-Henseleit buffer with or without 11 mmol/L glucose, followed by 32 minutes of reperfusion with buffer containing 11 mmol/L glucose. 31P nuclear magnetic resonance spectra were acquired sequentially throughout. Further experiments involved early reperfusion or the addition of HOE 694, a Na+-H+ exchange inhibitor, during reperfusion. When glucose was supplied throughout ischemia, no ischemic contracture occurred, and postischemic recovery of contractile function was highest, at 88% of preischemic function. In the absence of glucose, normal-glycogen hearts underwent ischemic contracture at 5 minutes, had an end-ischemic pH of 6.87, and recovered to 54%, whereas in high-glycogen hearts, contracture was delayed to 13 minutes, the end-ischemic pH was 6.61, and functional recovery decreased to 13%. Contracture onset coincided with the decrease in glycolysis, which occurred as glycogen became fully depleted. Functional recovery in the high-glycogen hearts increased to 89% when reperfused before contracture and to 56% when reperfused in the presence of HOE 694. Thus, during brief ischemia in the high-glycogen hearts, ischemic glycogen depletion and contracture were avoided, and the hearts were protected from injury. In contrast, during prolonged ischemia in the high-glycogen hearts, glycogen became fully depleted, and myocardial injury occurred; the injury was exacerbated by the lower ischemia pH in these hearts, leading to increased Na+-H+ exchange during reperfusion. The contradictory findings of past studies concerning the effect of high glycogen on the ischemic myocardium may thus be due to differences in the extent of glycogen depletion during ischemia.     

Overexpression of the cardiac Na+/Ca2+ exchanger increases susceptibility to ischemia/reperfusion injury in male, but not female, transgenic mice

Influx of Ca2+ into myocytes via Na+/Ca2+ exchange may be stimulated by the high levels of intracellular Na+ and the changes in membrane potential known to occur during ischemia/reperfusion. This increased influx could, in turn, lead to Ca2+ overload and injury. Overexpression of the cardiac Na+/Ca2+ exchanger therefore may increase susceptibility to ischemia/reperfusion injury. To test this hypothesis, the hearts of male and female transgenic mice, overexpressing the Na+/Ca2+ exchange protein, and hearts of their wild-type littermates, were perfused with Krebs-Henseleit buffer and subjected to 20 minutes of ischemia and 40 minutes of reperfusion. Preischemic left ventricular developed pressures and +dP/dtmax, as well as -dP/dtmin, were higher in the male transgenic hearts compared with wild-type, implying a role for Na+/Ca2+ exchange in the contraction, as well as the relaxation, phases of the cardiac beat. Postischemic function was lower in male transgenic than in male wild-type hearts (7+/-2% versus 32+/-6% of preischemic function), but there was no difference between female transgenic and female wild-type hearts, both at approximately 30% of preischemic function. To assess whether this male/female difference was due to female-specific hormones such as estrogen, the hearts of bilaterally ovariectomized and sham-operated transgenic females were subjected to the same protocol. The functional recoveries of ovariectomized female transgenic hearts were lower (17+/-3% of preischemic function) than those of wild-type and sham-operated transgenic females. The lower postischemic functional recovery in the male transgenic and female ovariectomized transgenic hearts correlated with lower recoveries of the energy metabolites, ATP and phosphocreatine, as measured by 31P nuclear magnetic resonance spectroscopy. Alternans were observed during reperfusion in male transgenic and female ovariectomized transgenic hearts only, consistent with intracellular Ca2+ overload. Western analyses showed that alterations in the expression of the Na+/Ca2+ exchange or L-type Ca2+ channel proteins were not responsible for the protection observed in the female transgenic hearts. In conclusion, in males, overexpression of the Na+/Ca2+ exchanger reduced postischemic recovery of both contractile function and energy metabolites, indicating that the Na+/Ca2+ exchanger may play a role in ischemia/reperfusion injury. From the studies of females, however, it appears that this exacerbation of ischemia/reperfusion injury by overexpression of the Na+/Ca2+ exchanger can be overcome partially by female-specific hormones such as estrogen.     

Ischemic preconditioning: effects on pH, Na and Ca in newborn rabbit hearts during Ischemia/Reperfusion

In adult hearts, ischemic preconditioning (PC) has been shown to decrease ischemia-induced changes in intracellular pH (pHi) and [Ca] ([Ca]i) and decrease associated injury. These results are consistent with the interpretation that PC decreases the stimulus for Na uptake via Na/H exchange, thereby decreasing intracellular Na (Nai) accumulation, and thus decreasing the change in force driving Na/Ca exchange, which otherwise contributes to ischemia-induced increases in [Ca]i. Given documented age-related differences in myocardial responses to ischemia, we tested the hypothesis that in newborn hearts, PC will diminish intracellular [H], Nai, and [Ca]i during ischemia/reperfusion. NMR was used to measure pHi, Nai, [Ca]i, ATP, and PCr in isolated newborn (4-7 days) rabbit hearts Langendorff-perfused with Krebs-Henseleit solution equilibrated with 95% O2/5% CO2 at 36+/-1 degrees C. Control hearts were perfused 30 min before initiating 40 min global ischemia followed by 40 min reperfusion. PC hearts were treated the same except four 5-min intervals of ischemia each followed by 10 min of perfusion which preceded global ischemia. At end ischemia, pHi was higher in PC than control hearts (6.31+/-0.03 v 5.83+/-0.05; P<0.05). Similarly, PC diminished Nai-accumulation during ischemia and reperfusion (P<0.05). Control Nai rose from 16.2+/-2.6 to 108.8+/-10.3 (mEq/kg dry weight) and recovered to 55.2+/-10.1 and the corresponding values for PC hearts were 25.6+/-6.2, 70.0+/-7.9 and 21.9+/-5.2. PC also improved [Ca]i recovery during reperfusion (P<0.05). Control [Ca]i rose from 418+/-43 to 1100+/-78 (nm/l) and recovered to 773+/-63, whereas in PC hearts the values were 382+/-40, 852+/-136 and 371+/-45, respectively. In addition, PC decreased coronary resistance during reperfusion (P<0.05) as reflected by lower perfusion pressures under constant flow conditions (65.9+/-1.5 v 56. 1+/-4.1 mmHg at end of reperfusion). Finally, PC improved recovery of left-ventricular developed pressure (LVDP-43.8+/-12.0 v 17.2+/-3. 0% of control; P<0.05) and diminished CK release (607+/-245 v 2432+/-639 IU/g dry weight; P<0.05) during reperfusion. The results are consistent with the hypothesis.     

Overexpression of MnSOD protects against myocardial ischemia/reperfusion injury in transgenic mice

Generation of free radicals upon reperfusion has been cited as one of the major causes of ischaemia/reperfusion injury. The following series of experiments was designed to study the effect of manganese superoxide dismutase (MnSOD) overexpression in transgenic mice on ischemia/reperfusion injury. A species of 1.4 kb human MnSOD mRNA was expressed, and a 325% increase in MnSOD activity was detected in the hearts of transgenic mice with no changes in the other antioxidant enzymes or heat shock proteins. Immunocytochemical study indicated an increased labeling of MnSOD mainly in the heart mitochondria of the transgenic mice. When these hearts were perfused as Langendorff preparations for 45 min after 35 min of global ischemia, the functional recovery of the hearts, expressed as heart rate x left ventricular developed pressure, was 52 +/- 4% in the transgenic hearts as compared to 31 +/- 4% in the non-transgenic hearts. This protection was accompanied by a significant decrease in lactate dehydrogenase release from the transgenic hearts. Overexpression of MnSOD limited the infarct size in vivo in a left coronary artery ligation model. Our results demonstrate that overexpression of MnSOD renders the heart more resistant to ischemia/reperfusion injury.     

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.     

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.     

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.     

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.     

Modulation of adenosine effects in attenuation of ischemia and reperfusion injury in rat heart

We investigated whether xanthine oxidase-derived superoxide radical generation could be modified by interfering with adenosine transport and metabolism in reducing myocardial injury during post-ischemic reperfusion. Isolated rat hearts perfused at constant pressure were subjected to 20 min of pretreatment with test agents, followed by 40 min global ischemia and 30 min reperfusion with or without test agents. In hearts treated with adenosine deaminase inhibitor, erythro 9-(2-hydroxy-3-nonyl) adenine (EHNA), alone or together with a selective nucleoside transport blocker, p-nitrobenzylthioinosine (NBMPR), the accumulated amount of O-2. was significantly reduced [10.2+/-0.97, 11.6+/-2.4, 8.1+/-0.51, respectively, v 31.6+/-2.1 (s. e.) nmol/wet g/30 min in ischemic control, P<0.01]. A positive correlation between O-2. and inosine release was observed in the initial 5 min of reperfusion in hearts treated with either EHNA or NBMPR ( r=0.475, P<0.05). Furthermore, the accumulated amount of LDH release showed positive correlation with that of O-2. among the same groups (r=0.474, P<0.05). Both EHNA and NBMPR had the cardioprotective effect on the recovery of left ventricular end-diastolic pressure (LVEDP), ATP repletion, and build up of endogenous adenosine. This study suggests that : (1) adenosine metabolism can be manipulated towards the formation of O-2. during reperfusion, and it has an important bearing on the cardiac recovery of ischemic myocardium, (2) the generation of O-2. is related to only inosine release during initial reperfusion.     

Protective effect of K(ATP) openers in ischemic rat hearts treated with a potassium cardioplegic solution

ATP-sensitive potassium channel (KATP) openers directly protect ischemic myocardium, which may make them useful for treating patients undergoing cardiopulmonary bypass, but whether high-potassium-containing cardioplegic solutions would inhibit their protective effects is not clear. We determined whether additional protection greater than that provided by cardioplegia could be found for KATP openers. We studied the effect of 10 microM cromakalim or BMS-180448 pretreatment (10 min before cardioplegia) on severity of ischemia in isolated rat hearts given normothermic or cold St. Thomas' cardioplegic solution (16 mM K+). After cardioplegic arrest, the hearts were subjected to 30-min (normothermic) or 150-min (hypothermic) global ischemia, each followed by 30-min reperfusion. The cardioplegic solutions significantly protected the hearts, as measured by increased time to onset of contracture, enhanced recovery of function, and reduced lactate dehydrogenase (LDH) release. Cromakalim and BMS-180448 both further significantly increased time to contracture in both normothermic and hypothermic arrested hearts; this was accompanied by enhanced recovery of reperfusion contractile function and reduced cumulative LDH release. This additional protective effect of the K ATP openers was abolished by glyburide. Because administration of the K ATP openers only with the cardioplegic solution (1 min before global ischemia) was not efficacious, >1-min pretreatment apparently is necessary. K ATP openers provide additional protection to that afforded by cold or normothermic potassium cardioplegia in rat heart, although the timing of treatment may be crucial.     

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.     

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.     

Effect of protein kinase C inhibitors on cardioprotection by ischemic preconditioning depends on the number of preconditioning episodes

OBJECTIVES: This study examined the possibility that the role of PKC in [corrected] PC, and thus the response to PFC inhibitors, may differ depending on how many ischemic episodes are employed to precondition the heart. METHODS: In the first series of experiments, myocardial infarct was induced by 30 min of coronary occlusion and 3 h of reperfusion in the rabbit. Infarct size was determined by tetrazolium staining and expressed as a percentage of area at risk (%IS/AR). Prior to the 30-min ischemia, rabbits were subjected to no PC, single PC (i.e., PC with an episode of 5 min ischemia/5 min reperfusion), and repetitive PC (2 cycles of 5 min ischemia/5 min reperfusion) with or without one of three treatments: polymyxin B (PolyB), staurosporine (Stauro), and 8-sulfophenylthephylline (SPT). In the second series of experiments, the rabbits received 5 min of coronary occlusion after repetitive PC with or without PolyB or Stauro treatment. Then, myocardial tissue in the ischemic region was sampled for assay of PKC activity. Untreated rabbits served as controls. RESULTS: Single and repetitive PC limited %IS/AR to the same extent (%IS/AR = 9.8 +/- 1.9 and 10.4 +/- 2.3, both p < 0.05, vs. the control value of 44.5 +/- 3.4), and single PC was blocked by PolyB (%IS/AR = 43.9 +/- 2.7) and Stauro (%IS/AR = 31.5 +/- 3.2). Although the protocol of PolyB injection maintained the plasma PolyB level during sustained ischemia well above its Ki for PKC, this agent and also Stauro failed to abolish the protection by repetitive PC (%IS/AR = 21.6 +/- 3.0 and 11.4 +/- 4.3, respectively). SPT, an adenosine receptor antagonist, not only blocked single PC (%IS/AR = 44.4 +/- 4.4) but also attenuated protection by repetitive PC (%IS/AR = 28.3 +/- 3.6). Infarct sizes in non-preconditioned hearts were not modified by PolyB, Stauro, or SPT. The ratio of membrane fraction PKC activity to cytosolic fraction PKC activity was elevated by repetitive PC plus 5 min ischemia, and this change in PKC was inhibited in hearts given PolyB and Stauro. CONCLUSIONS: In contrast to single PC, repetitive PC protects the heart against infarction even when PolyB and Stauro are administered to inhibit PKC during ischemic insult. This difference may be attributable to a PKC-independent mechanism, in which the adenosine receptor may be partly involved.     

Dichloroacetate enhanced myocardial functional recovery post-ischemia : ATP and NADH recovery

This study was undertaken to determine the effect of dichloroacetate (DCA) on myocardial functional and metabolic recovery following global ischemia. Isolated rabbit hearts were subjected to 120 min of mildly hypothermic (34 degrees C), cardioplegic arrest with multidose, modified St. Thomas' cardioplegia. Hearts were reperfused with either physiologic salt solution (PSS) as controls, (CON, n = 10) or PSS containing DCA (DCA, n = 6) at a concentration of 1 mM. Functional and metabolic indices were determined at baseline and at 15, 30, and 45 min of reperfusion. In four DCA and four CON hearts, myocardial biopsies were taken at baseline, end-ischemia, 15 and 45 min for nucleotide levels. Functional recovery was significantly better in hearts reperfused with DCA as demonstrated by recovery of baseline developed pressure (DCA = 69 +/- 5%, CON = 45 +/- 9%) and dP/dt (DCA = 64% +/- 10% versus CON = 48% +/- 10%). Coronary blood flow was not different between groups either at baseline or during reperfusion, but myocardial oxygen consumption (MVO2) was increased in the DCA versus CON hearts (79% +/- 20% of baseline vs 50% +/- 18%). Recovery of myocardial adenylate energy status was improved in the DCA versus CON hearts (ATP recovered to 45% +/- 20% versus 8% +/- 6% of baseline). Coronary sinus lactate concentration was decreased in DCA perfused hearts at 45 min of reperfusion. Percent of baseline NADH values was similar at 15 min of reperfusion, but at 45 min, DCA hearts showed a decrease in NADH levels, while CON hearts showed an increase (DCA = 48%; CON = 121%). The enhanced myocardial function and improved metabolic status noted with DCA may result from increased oxidative phosphorylation due to altered pyruvate dehydrogenase (PDH) activity.     

Generalized peroxisomal disorder in male twins: fatty acid composition of serum lipids and response to n-3 fatty acids

Ischemia leads to intracellular acidification which can be counteracted by the Na+/H+-exchange mechanism. A blockade of this exchanger has been hypothesized to cause stronger intracellular acidification in the course of ischemia thereby protecting the heart from ischemic damage. The aim of our study was to find out (1) whether in the course of ischemia areas become electrically silent, (2) whether this is enhanced by the Na+/H+-exchange inhibitor cariporide (4-Isopropyl-3-methylsulfonylbenzoyl-guanidine; Hoe 642) and whether cariporide has protective effects. Therefore, we submitted isolated rabbit hearts, perfused according to the Langendorff technique to regional ischemia (LAD occlusion) for 30 min followed by 30 min reperfusion with (n=7) or without (n=7) pre-treatment with 1 microM cariporide. Under these conditions 256-channel epicardial potential mapping was carried out. Under non-ischemic conditions cariporide did not alter any of the parameters under observation. We found that ischemia led to marked alterations of the activation pattern, to action potential shortening and a marked increase in the dispersion of refractoriness. In the ischemic region there was a significant ST deviation from the isoelectrical line (control 32+/-10; 30 min ischemia: 290+/-35 arbitrary units [a.u.]). This was markedly reduced by cariporide (control 39+/-10; 30 min ischemia: 170+/-25 a.u.). The increase in dispersion by ischemia (by 50+/-5 ms) was significantly counteracted by cariporide (increased dispersion by 20+/-4 ms). In a similar way the alteration of the activation pattern was antagonized. Under the influence of cariporide we found a lower increase in the left ventricular enddiastolic pressure, and a significantly slower recovery of the action potential duration. After 30 min of ischemia 24+/-5 (control series) 24.5+/-5 mm2 (cariporide) became electrically silent. In a second series of experiments the incidence of arrhythmia was assessed: we found ventricular fibrillation in 6/7 untreated control hearts and in 4/7 cariporide treated hearts. In a third series of experiments we determined the intracellular [ATP] after 30 min of LAD occlusion using a histochemical method. We observed a decrease in [ATP] in the ischemic region as compared to the non-ischemic right ventricular wall, which was less pronounced in cariporide-treated hearts. Thus, we conclude that (1) cariporide protects the heart from ischemic damage and (2) at least under these conditions an enlargement of the electrically silent area did not occur.     

NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline) did not affect recovery of high energy phosphates and pH in early reperfusion in a rat model of transient forebrain ischemia. Or: an in vivo 31P NMR spectroscopy study

The new non-NMDA (N-methyl-D-aspartate) receptor antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline) has previously been shown to exert a neuroprotective effect in animal models of cerebral ischemia when administered in the post-ischemic phase. In this investigation the effect of NBQX on acidosis and energy recovery in early reperfusion after 10 min of transient forebrain ischemia with the 2-vessel occlusion model in the rat was studied with 31P NMR spectroscopy. In the intervention group the animals received a bolus dose of NBQX 30 mg.kg-1 i.v. at the start of reperfusion. 31P NMR spectroscopy was used to measure intracellular pH, ATP and phosphocreatine continuously in-vivo during, and after, the ischemic event. The recovery of high energy phosphates and pH was followed during 30 min of reperfusion. Pre-ischemic levels of phosphocreatine were reached after approximately 9-10 min in both groups. Although a slight improvement could be seen in the intervention group there was no significant difference in the rate of recovery between the two groups. ATP reached 90% of preischemic levels after about 8 min without significant difference between the two groups. With respect to the recovery of intracellular pH, no difference could be shown. Our results do not contradict previously published results, but suggest that the potential protective effect of NBQX is not mediated through improved recovery of energy metabolism in early reperfusion.     

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.     

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.     

Developmental differences in superoxide production in isolated guinea-pig hearts during reperfusion

The production of free radicals on reperfusion has been implicated as an important factor governing post-ischemic recovery of cardiac function. Although the response of the heart to ischemia and reperfusion is known to change during cardiac development, it is not known if different rates of free radical production play a role in these altered responses. The aim of this investigation was to determine if the production of the superoxide anion (O2-) on reperfusion differs in the immature and mature heart. Immature hearts, obtained from 3-day premature guinea pigs (delivered by cesarean section) were compared with those from adults (7 weeks old). Using the isolated Langendorff preparation. O2- production was measured during reperfusion following ischemic durations [0 (aerobic control), 15, 20, 30, and 60 min, n = 6/group] by the reduction of succinylated ferricytochrome c in the perfusate. Both immature and mature hearts exhibited bell-shaped relationship between ischemic duration and peak O2- production on reperfusion: (13.4 +/- 5.9; 22.2 +/- 5.4; 23.0 +/- 7.8; 59.3 +/- 16.2; 33.7 +/- 15.1; 32.6 +/- 8.5 nmol/min/g wet weight in the immature heart and 15.7 +/- 1.9; 55.0 +/- 30.2; 82.8 +/- 14.0; 78.8 +/- 33.8; 40.6 +/- 16.4; 45.4 +/- 13.1 nmol/min/g wet weight in the mature heart after 0; 15; 20; 30; 45 and 60 min of ischemia, respectively). A similar relationship was also demonstrated with O2- production over the 20-min reperfusion period: (134.0 +/- 57.1; 106.5 +/- 46.2; 199.3 +/- 50.6; 362.0 +/- 99.5; 375.0 +/- 60.9; 221.0 +/- 73.0 nmol/20 min/g wet weight in the immature heart and 97.8 +/- 54; 282.0 +/- 139.0; 933.3 +/- 210.3; 964.0 +/- 374.0; 443.0 +/- 106.0; 352.0 +/- 1551.0 nmol/20 min/g wet weight in the mature heart after 0, 15, 20, 30, 45 and 60 min of ischemia, respectively). Mature hearts consistently produced more O2- than immature hearts on reperfusion, while there was no significant difference in their capacity to produce O2- during aerobic perfusion. We conclude that the immature heart may be at less risk from the free radical component of reperfusion injury than the mature heart.