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Recovery from severe immunosuppression requires hematopoietic stem cell reconstitution and effective thymopoiesis to restore a functional immune cell repertoire. Herein, a model of immune cell reconstitution consequent to potentially lethal doses of irradiation is described, which may be valuable in evaluating potential medical countermeasures. Male rhesus macaques were total body irradiated by exposure to 6.00 Gy 250 kVp x-radiation (midline tissue dose, 0.13 Gy min), resulting in an approximate LD10/60 (n = 5/59). Animals received medical management, and hematopoietic and immune cell recovery was assessed (n ≤ 14) through 370 d post exposure. A subset of animals (n ≤ 8) was examined through 700 d. Myeloid recovery was assessed by neutrophil and platelet-related parameters. Lymphoid recovery was assessed by the absolute lymphocyte count and FACS-based phenotyping of B- and T-cell subsets. Recent thymic emigrants were identified by T cell receptor excision circle quantification. Severe neutropenia, lymphopenia, and thrombocytopenia resolved within 30 d. Total CD3+ cells μL required 60 d to reach values 60% of normal, followed by subsequent slow recovery to approximately normal by 180 d post irradiation. Recovery of CD3+4+ and CD3+8+ cell memory and naïve subsets were markedly different. Memory populations were ≥ 100% of normal by day 60, whereas naïve populations were only 57% normal at 180 d and never fully recovered to baseline post irradiation. Total (CD20+) B cells μL were within normal levels by 77 d post exposure. This animal model elucidates the variable T- and B-cell subset recovery kinetics after a potentially lethal dose of total-body irradiation that are dependent on marrow-derived stem and progenitor cell recovery, peripheral homeostatic expansion, and thymopoiesis.

The development of medical countermeasures against the hematopoietic subsyndrome of the acute radiation syndrome requires well characterized and validated animal models. The model must define the radiation dose- and time-dependent relationships for mortality and major signs of morbidity to include other organ damage that may contribute to morbidity and mortality. Herein, the authors define these parameters for a nonhuman primate exposed to total body radiation and administered medical management. A blinded, randomized study (n = 48 rhesus macaques) determined the lethal dose-response relationship using bilateral 6 MV linear accelerator photon radiation to doses in the range of 7.20 to 8.90 Gy at 0.80 Gy min(-1). Following irradiation, animals were monitored for complete bloodcounts, body weight, temperature, diarrhea, and hydration status for 60 d. Animals were administered medical management consisting of intravenous fluids, prophylactic antibiotics, blood transfusions, anti-diarrheals, analgesics, and nutrition. The primary endpoint was survival at 60 d post-irradiation; secondary endpoints included hematopoietic-related parameters, number of transfusions, incidence of documented infection, febrile neutropenia, severity of diarrhea, mean survival time of decedents, and tissue histology. The study defined an LD30/60 of 7.06 Gy, LD50/60 of 7.52 Gy, and an LD70/60 of 7.99 Gy with a relatively steep slope of 1.13 probits per linear dose. This study establishes a rhesus macaque model of the hematopoietic acute radiation syndrome and shows the marked effect of medical management on increased survival and overall mean survival time for decedents. Furthermore, following a nuclear terrorist event, medical management may be the only treatment administered at its optimal schedule.

Pre-ischemic treatment is seldom possible in the clinical setting of acute myocardial infarction. Thus, to successfully save myocardium from infarction, it is required that protective interventions must be effective when applied after ischemia has begun or at the onset of reperfusion. Unfortunately, in spite of a large body of experimental data showing that various interventions are cardioprotective at reperfusion, no specific therapy has yet been established to be clinically applicable. However, recent data from several laboratories have shown that adenosine and its analogues given at reperfusion can markedly protect the heart from ischemia/reperfusion injury. While the experimental data suggest that factors such as adenosine A2 receptor activation, anti-neutrophil effect, attenuation of free radical generation, increased nitric oxide (NO) availability, activation of the PI3-kinase/Akt pathway and ERK, prevention of mitochondrial damage, and anti-apoptotic effects may be involved in the protective effect of adenosine or its analogues, the exact receptor subtype(s), the detailed signaling mechanisms, and interaction between those individual factors are still unknown. A definite answer to these unsolved problems will offer insights into the mechanisms of cardioprotection at reperfusion, and will be critical for developing a successful therapeutic strategy to salvage ischemic myocardium in patients with acute myocardial infarction.

Ischemic and pharmacological preconditioning can be triggered by an intracellular signaling pathway in which Gi-coupled surface receptors activate a cascade including phosphatidylinositol 3-kinase, endothelial nitric oxide synthase, guanylyl cyclase, and protein kinase G (PKG). Activated PKG opens mitochondrial KATP channels (mitoKATP) which increase production of reactive oxygen species. Steps between PKG and mitoKATP opening are unknown. We describe effects of adding purified PKG and cGMP on K+ transport in isolated mitochondria. Light scattering and respiration measurements indicate PKG induces opening of mitoKATP similar to KATP channel openers like diazoxide and cromakalim in heart, liver, and brain mitochondria. This effect was blocked by mitoKATP inhibitors 5-hydroxydecanoate, tetraphenylphosphonium, and glibenclamide, PKG-selective inhibitor KT5823, and protein kinase C (PKC) inhibitors chelerythrine, Ro318220, and PKC-epsilon peptide antagonist epsilonV(1-2). MitoKATP are opened by the PKC activator 12-phorbol 13-myristate acetate. We conclude PKG is the terminal cytosolic component of the trigger pathway; it transmits the cardioprotective signal from cytosol to inner mitochondrial membrane by a pathway that includes PKC-epsilon.

CGX-1051, isolated from the venom of the marine snail Conus purpurasens, was previously noted to interact with potassium channels. Since potassium channels play an important role in cardiac physiology, we assessed the effect of CGX-1051 on infarct size in a rabbit heart model of ischemia/reperfusion. A coronary branch was occluded for 30 minutes followed by 3 hours of reperfusion in in situ and 2 hours in in vitro preparations. Infarct size was measured with triphenyltetrazolium chloride staining and expressed as a percent of the risk zone. In in situ studies, a bolus intravenous injection of CGX-1051, either 10 or 100 microg/kg, administered 5 minutes before reperfusion, reduced infarct size from 40.4 +/- 2.8% of the risk zone in untreated animals to 19.8 +/- 3.8% and 15.0 +/- 1.9%, respectively. One microg/kg CGX-1051 was not protective. To see if the salvage was sustained, two groups of rabbits underwent 72 hours of reperfusion. The dose of 10 microg/kg infused 5 minutes before reperfusion reduced infarct size from 37.0 +/- 1.6% in untreated rabbits to 15.5 +/- 2.0%. When administered 10 minutes after reperfusion had begun, 100 microg/kg CGX-1051 had no effect. CGX-1051 also reduced infarct size in crystalloid-perfused, isolated rabbit hearts suggesting that protection did not depend on circulating leukocytes. The mitochondrial KATP inhibitors glibenclamide and 5-hydroxydecanoate and the MEK(1/2), ERK and hence, inhibitor PD 98059 aborted protection from CGX-1051. These data indicate that functionally active ERK and mitochondrial KATP channels are necessary for protection. CGX-1051 caused no hemodynamic alterations at any dose tested. We conclude that CGX-1051 has a powerful anti-infarct effect when given just before reperfusion.

The adenosine A(1)/A(2) receptor agonist AMP 579 has been reported to protect the heart against infarction even when administered after the onset of ischemia. The present study explored both the timing and the duration of treatment required to limit infarct size in in situ rabbit hearts subjected to 30 min of regional ischemia and 3 hours of reperfusion. In groups 1 and 2, AMP 579 was infused from 10 min before reperfusion and continued for either 30 or 40 min. In group 3, AMP 579 was begun at the onset of reperfusion and continued for 70 min. In group 4, AMP 579 was also infused for 70 min but begun 10 min after reperfusion. In untreated hearts 36.4 +/- 3.1% of the risk zone infarcted. Protection was observed only in hearts having a 70-minute infusion of AMP 579 starting at reperfusion (13.0 +/- 1.9%, P < 0.05). Therefore, AMP 579 must be present at the moment of reperfusion and have a continued presence of more than 30 min thereafter to protect. Importantly, because AMP 579 can protect when administered up to the time of reperfusion, it likely prevents a reperfusion injury, and, therefore, has impressive clinical potential.

AMP 579, an adenosine A /A receptor agonist, is cardioprotective when administered at reperfusion. Pretreatment with the Na /H exchanger inhibitor cariporide or ischemic preconditioning (PC) also limits infarct size. To gain insight into the mechanism of AMP 579 we investigated whether its protection could be added to that from either cariporide or PC. rabbit hearts were subjected to 45 min of regional ischemia followed by 3 h of reperfusion. Infarct size in the control group was 55.8 +/- 3.9% of the risk zone. PC significantly reduced infarct size to 26.0 +/- 6.7% (p<0.05). AMP 579 (30 micro g/kg) given just before reperfusion followed by 3 micro g/kg/min infusion for 70 min also limited infarct size (32.1 +/- 1.8%,) but the combination of AMP 579 and PC showed a significantly greater limitation of infarct size (5.5 +/- 2.7%, p < 0.05). Because cariporide pretreatment was so protective (8.5 +/- 3.7% infarction), we had to increase the ischemic insult to 60 min to test for any additive effect of the combination of AMP 579 + cariporide. Infarct size in the untreated group was 66.0 +/- 4.9% of the risk zone. Cariporide (0.5 mg/kg) 5 min prior to ischemia significantly reduced infarct size to 41.5 +/- 7.7%. When cariporide pre-treatment was combined with AMP 579 at reperfusion, infarction was further limited (14.2 +/- 4.5%). Because AMP 579's protection can be added to that of either cariporide or PC, AMP 579's mechanism of protection probably differs from either of them. The combination of AMP 579 + cariporide was particularly efficacious and could be useful in the surgical setting.

In the rabbit heart, acute ethanol exposure followed by washout before ischemia exerts a preconditioninglike effect. However, if alcohol is still present during ischemia, all preconditioning-related cardioprotection is abolished. The present follow-up study investigated the dose-response relationships of both the beneficial and detrimental effects of acute ethanol exposure. In the isolated rabbit heart either 2.5-, 5-, 10-, 20-, or 50-mmol/L ethanol was given as a 5-minute pulse followed by washout before 30 minutes of regional ischemia and 2 hours of reperfusion. Isolated rabbit hearts were also preconditioned with 5 minutes of global ischemia followed by 10 minutes of reperfusion (PC) before onset of 30 minutes of regional ischemia. This latter protocol was combined with a 35-minute infusion of ethanol at concentrations of either 5, 10, 20, or 50 mmol/L, starting 5 minutes before the onset of the 30-minute period of ischemia. Infarct size was determined with triphenyltetrazolium staining. No protection was seen with a 5-minute infusion of 2.5-mmol/L ethanol (29.9 +/- 1.6% of risk zone infarcted), and minimal protection was evident with the 5-mmol/L dose (25.4 +/- 3.4% infarction). In all other groups infarct size was significantly reduced (17.9 +/- 3.2, 18.4 +/- 3.5, and 16.8 +/- 3.4%, respectively, versus 33.0 +/- 3.0% in control group, P < 0.05). In the presence of 10-, 20-, or 50-mmol/L ethanol, infarct size following PC was not different from control (24.3 +/- 2.5, 28.4 +/- 4.3, and 39.0 +/- 4.0%, respectively, versus 28.5 +/- 2.5%). Thus the presence of alcohol during ischemia inhibited protection induced by preceding preconditioning ischemia. Only in the PC group exposed to 5-mmol/L ethanol was infarct size significantly smaller than in the control group (6.4 +/- 2.5%, P < 0.005). Thus both protective and antiprotective effects of alcohol were dose dependent with similarly low threshold doses in in vitro rabbit hearts. Since it might be impossible to find a dose of ethanol that would be protective if administered shortly before ischemia, ethanol should be removed before that ischemia to protect myocardium.

To determine the mechanism by which AMP 579, an adenosine A1/A2 agonist, administered at reperfusion protects ischemic myocardium, buffer-perfused rabbit hearts were subjected to 30 min of global ischemia and 2 h of reperfusion. AMP 579 (500 nM) was included in the reperfusate for the first 70 min. Average left ventricular diastolic pressure during reperfusion in hearts receiving AMP 579 was lower than that in control hearts (17.9 +/- 2.4 vs. 39.0 +/- 6.5 mm Hg, p < 0.05), indicating attenuation of contracture. Left ventricular developed pressure and coronary flow during reperfusion were also significantly improved with AMP 579 treatment. AMP 579's anti-contracture effect was blocked by the adenosine A2-receptor antagonist 8-(3-chlorostyryl)caffeine (CSC), but not by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). CSC, but not DPCPX, also blocked AMP 579's ability to preserve developed pressure and coronary flow in these hearts. AMP 579 significantly reduced infarction in isolated hearts subjected to regional ischemia. The anti-infarct effect again was abolished by CSC but not by DPCPX. Finally, we tested whether 5'-(N-ethylcarboxamido)adenosine (NECA), another A1/A2 agonist, also administered for the initial 70 min of reperfusion, could duplicate the anti-infarct effect of AMP 579. One-hundred-nanomolar NECA duplicated the protection, but neither 50 nM CGS21680, a selective A2 agonist, nor 100 microM adenosine was protective. Therefore, AMP 579 given at reperfusion reduces contracture and infarction. Anti-contracture and anti-infarct effects require the adenosine A2, but not the A1, receptor suggesting that prevention of contracture and tissue salvage are mechanistically related. Not all A2 agonists were able to duplicate the anti-infarct effect, suggesting something unique about AMP579.

It has been assumed that all G(i)-coupled receptors trigger the protective action of preconditioning by means of an identical intracellular signaling pathway. To test this assumption, rabbit hearts were isolated and perfused with Krebs buffer. All hearts were subjected to a 30-minute coronary artery occlusion followed by 120 minutes of reperfusion. Risk area was measured with fluorescent particles and infarct size with triphenyltetrazolium chloride staining. Control hearts showed 29.1+/-2.8% infarction of the risk zone. A 5-minute infusion of acetylcholine (0.55 mmol/L) beginning 15 minutes before the 30-minute occlusion resulted in significant protection (9.2+/-2.7% infarction). This protection could be blocked by administration of 300 micromol/L N-2-mercaptopropionyl glycine (MPG), a free radical scavenger, or by 200 micromol/L 5-hydroxydecanoate (5-HD), a mitochondrial K(ATP) antagonist, for 15 minutes beginning 5 minutes before the acetylcholine infusion (35.2+/-3.9% and 27.8+/-2.4% infarction, respectively). Similar protection was observed with other known triggers, ie, bradykinin (0.4 micromol/L), morphine (0.3 micromol/L), and phenylephrine (0.1 micromol/L), and in each case protection was completely abrogated by either MPG or 5-HD. In contrast, protection by adenosine or its analog N(6)-(2-phenylisopropyl) adenosine could not be blocked by either MPG or 5-HD. Therefore, whereas most of the tested agonists trigger protection by a pathway that requires opening of mitochondrial K(ATP) channels and production of free radicals, the protective action of adenosine is not dependent on either of these steps. Hence, it cannot be assumed that all G(i)-coupled receptors use the same signal transduction pathways to trigger preconditioning.

The critical time for opening mitochondrial (mito) K(ATP) channels, putative end effectors of ischemic preconditioning (PC), was examined. In isolated rabbit hearts 29+/-3% of risk zone infarcted after 30 minutes of regional ischemia. Ischemic PC or 5-minute exposure to 10 micromol/L diazoxide, a mito K(ATP) channel opener, reduced infarction to 3+/-1% and 8+/-1%, respectively. The mito K(ATP) channel closer 5-hydroxydecanoate (200 micromol/L), bracketing either 5-minute PC ischemia or diazoxide infusion, blocked protection (24+/-3 and 28+/-6% infarction, respectively). However, 5-hydroxydecanoate starting 5 minutes before long ischemia did not affect protection. Glibenclamide (5 micromol/L), another K(ATP) channel closer, blocked the protection by PC only when administered early. These data suggest that K(ATP) channel opening triggers protection but is not the final step. Five minutes of diazoxide followed by a 30-minute washout still reduced infarct size (8+/-3%), implying memory as seen with other PC triggers. The protection by diazoxide was not blocked by 5 micromol/L chelerythrine, a protein kinase C antagonist, given either to bracket diazoxide infusion or just before the index ischemia. Bracketing preischemic exposure to diazoxide with 50 micromol/L genistein, a tyrosine kinase antagonist, did not affect infarction, but genistein blocked the protection by diazoxide when administered shortly before the index ischemia. Thus, although it is not protein kinase C-dependent, the protection by diazoxide involves tyrosine kinase. Bracketing diazoxide perfusion with N:-(2-mercaptopropionyl) glycine (300 micromol/L) or Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (7 micromol/L), each of which is a free radical scavenger, blocked protection, indicating that diazoxide triggers protection through free radicals. Therefore, mito K(ATP) channels are not the end effectors of protection, but rather their opening before ischemia generates free radicals that trigger entrance into a preconditioned state and activation of kinases.

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

Recent studies suggest that p38 mitogen-activated protein kinase (MAPK) may be involved in ischemic preconditioning (PC). To further test this possibility, the regulation of MAPK-activated protein kinase 2 (MAPKAPK2), a kinase immediately downstream from p38 MAPK, and the activity of c-Jun NH(2)-terminal kinase (JNK), a second MAPK, were examined in preconditioned hearts. Isolated, perfused rabbit hearts were subjected to 20 to 30 minutes of global ischemia. Ventricular biopsies before treatment and after 20 minutes of ischemia were homogenized, and the activities of MAPKAPK2 and JNK were evaluated. For the MAPKAPK2 experiments, 7 groups were studied, as follows: control hearts; preconditioned hearts; hearts treated with 500 nmol/L R(-) N(6)-(2-phenylisopropyl) adenosine (PIA), an A(1)-adenosine receptor agonist; preconditioned hearts pretreated with 100 micromol/L 8-(p-sulfophenyl) theophylline (SPT), an adenosine receptor antagonist; preconditioned hearts also treated with SB 203580, a potent inhibitor of p38 MAPK activation; hearts treated with 50 ng/mL anisomycin (a p38 MAPK/JNK activator); and hearts treated with both anisomycin (50 ng/mL) and the tyrosine kinase inhibitor genistein (50 micromol/L). MAPKAPK2 activity was not altered in control hearts after 20 minutes of global ischemia. By contrast, there was a 3.8-fold increase in activity during ischemia in preconditioned hearts. Activation of MAPKAPK2 in preconditioned hearts was blocked by both SPT and SB 203580. MAPKAPK2 activity during ischemia increased 3.5-fold and 3.3-fold in hearts pretreated with PIA or anisomycin, respectively. MAPKAPK2 activation during ischemia in hearts pretreated with anisomycin was blocked by genistein. In separate hearts, anisomycin mimicked the anti-infarct effect of PC, and that protection was abolished by genistein. JNK activity was measured in control and preconditioned hearts. There was a comparable, modest decline in activity during 30 minutes of global ischemia in both groups. As a positive control, a third group of hearts was treated with anisomycin before global ischemia, and in these, JNK activity increased by 290% above baseline. These results confirm that the p38 MAPK/MAPKAPK2 pathway is activated during ischemia only if the heart is in a preconditioned state. These data further support p38 MAPK as an important signaling component in ischemic PC.

Background: The role of protein phosphatases (PPs) during ischemic preconditioning in the rabbit heart was examined., Methods and Results: Fostriecin, a potent inhibitor of PP2A, was administered to isolated rabbit hearts starting either 15 minutes before or 10 minutes after the onset of a 30-minute period of regional ischemia and continuing until the onset of reperfusion. After 2 hours of reperfusion, infarct size was measured with triphenyltetrazolium chloride. In a second study with isolated rabbit cardiomyocytes, the effect of fostriecin pretreatment was assessed by measuring changes in cell osmotic fragility during simulated ischemia. PP1 and PP2A activities of isolated control and ischemically preconditioned cells were also measured. In a third series of experiments, left ventricular biopsies of isolated rabbit hearts were obtained before and at selected times during 60 minutes of global ischemia, and the tissue was assayed for PP1 and PP2A activities. In isolated hearts pretreated with fostriecin, only 8% of the ischemic zone infarcted, significantly less than that in untreated control hearts (33%; P<0.001) but comparable to that in ischemically preconditioned hearts (9%; P<0.001 versus control). Significant protection was also observed in the hearts treated only after the onset of ischemia (18% infarction; P<0.05 versus control). In isolated myocytes, fostriecin also provided protection comparable to that produced by metabolic preconditioning. Preconditioning had no apparent effect on the activity of either PP1 or PP2A in isolated ventricular myocytes or ventricular tissue obtained from heart biopsies., Conclusions: Fostriecin, a potent inhibitor of PP2A, can protect the rabbit heart from infarction even when administered after the onset of ischemia. But inhibition of either PP1 or PP2A does not appear to be the mechanism of protection from ischemic preconditioning.

Background: The influence of anesthetic agents on the infarction process in the ischemic myocardium is unclear. This study evaluated the effects of three intravenous and three inhalational anesthetic agents on myocardial infarction within a quantified ischemic risk zone in rabbit hearts subjected to a standardized regional ischemia-reperfusion insult., Methods: Both in vitro and in situ rabbit models were used to investigate the effects of anesthetic agents on infarct size. In all rabbits the heart was exposed and a coronary artery surrounded with a suture to form a snare for subsequent occlusion. In in situ preparations, both intravenous and inhalational agents were tested, whereas only the latter were used in isolated hearts. Infarct size was determined by triphenyltetrazolium chloride staining. To determine whether an adenosine-mediated protective mechanism was involved, 8-(p-sulfophenyl)theophylline, an adenosine receptor blocker, was added to halothane-treated isolated hearts. Adenosine concentration in the coronary effluent was also measured in isolated hearts exposed to halothane. In other protocols, chelerythrine, a highly selective protein kinase C inhibitor, was administered to both halothane-treated and untreated isolated hearts., Results: Infarcts in the three in situ groups anesthetized with halothane, enflurane, and isoflurane were about one half as large as infarcts in rabbits that underwent anesthesia with pentobarbital, ketamine-xylazine, or propofol. Volatile anesthetics also protected isolated hearts by a similar amount. Both adenosine receptor blockade and chelerythrine abolished cardioprotection from halothane in isolated hearts. Halothane treatment did not increase adenosine release., Conclusions: The volatile anesthetics tested protected the ischemic rabbit heart from infarction, in contrast to the three intravenous agents tested. Protection was independent of the hypotensive effect of the inhalational agents because halothane also protected isolated hearts, in which changing vascular tone is not an issue and coronary perfusion pressure is constant. Cardioprotection by volatile anesthetics depended on both adenosine receptors and protein kinase C, and thus is similar to the mechanism of protection seen with ischemic preconditioning.

Background: Activation of protein kinase C (PKC) is thought to be a critical step in ischemic preconditioning. Many receptor agonists activate PKC via stimulation of phospholipase C (PLC), which degrades membrane phospholipids to diacylglycerol (DAG), an important PKC cofactor. However, adenosine receptors, critical components of the prototypical preconditioning pathway, are not thought to couple to PLC in the cardiomyocyte. We therefore tested whether ischemic preconditioning or adenosine might instead activate phospholipase D (PLD) to produce DAG., Methods and Results: PLD activity was measured in isolated rabbit hearts. Ischemic injury was evaluated in either isolated rabbit hearts or dispersed myocytes. PLD activity doubled from a control level of 74.8 +/- 10.0 to 140.0 +/- 11.5 mumol.min-1.g-1 (P < .025) after two 5-minute periods of global ischemia separated by 5 minutes of reperfusion. A similar increase was noted after the heart had been exposed to (R)-N6-(2-phenylisopropyl)-adenosine [(R)-PIA] for 20 minutes. When sodium oleate, which activates PLD, was administered to isolated hearts before a 30-minute coronary occlusion, infarct size (15.6 +/- 2.0% of the risk zone) was significantly smaller than in untreated hearts (30.4 +/- 2.2%; P < .01). Exposure to sodium oleate significantly prolonged the rate of isolated myocyte survival during simulated ischemia. Propranolol 100 mumol/L, which blocks DAG production from metabolites produced by PLD catalysis, completely abolished the protective effects of both metabolic preconditioning and (R)-PIA exposure in myocytes., Conclusions: We conclude that PLD stimulation is involved in the protection of ischemic preconditioning in the rabbit heart.

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

Ischemic preconditioning in the rabbit is initiated by adenosine A1-receptor stimulation, which activates protein kinase C (PKC). Additionally, alpha 1-adrenergic agonists can similarly protect ischemic myocardium, but there has been confusion about the role adenosine receptors play in this protection. To characterize the interaction between adrenergic and adenosine receptors and to study the possible role of PKC in this protection, we used isolated rabbit hearts perfused with oxygenated Krebs' buffer. All hearts were subjected to 30 minutes of regional myocardial ischemia and 2 hours of reperfusion. Infarct size was determined by triphenyltetrazolium staining. Pharmacologic preconditioning in hearts with a 5-minute phenylephrine (PE) infusion 10 minutes before the prolonged regional ischemia resulted in significantly smaller infarcts (9.7 +/- 1.3% of risk area) than in control hearts (31.0 +/- 2.6%, P < .05). This protection could be effectively blocked by administration of the alpha-adrenergic blocker phenoxybenzamine. Methoxamine, an alpha 1a-selective agonist, failed to protect, whereas the alpha 1b-selective antagonist chloroethylclonidine aborted the protective effect of PE. Polymyxin B, an inhibitor of PKC, also blocked the protective effect of PE, implying that PKC has an important role in preconditioning. The adenosine receptor blocker 8-(p-sulfophenyl)theophylline (SPT) given at the same time as the PE infusion did not affect the protection, implying that an alpha 1-agonist could initiate protection independent of adenosine, presumably by direct coupling to PKC. However, the protective effect of PE could be blocked if SPT were administered during the 30-minute regional ischemia. This observation suggested that adenosine receptor occupancy is necessary during long ischemia to reactivate PKC and mediate the protection. However, the addition of a second PE infusion beginning 5 minutes before and continuing throughout the long ischemic period restored the protective effect of PE despite the presence of SPT. Thus, as long as at least one of the receptors (alpha 1-adrenegic or adenosine A1) is activated during long ischemia, protection will be realized. These data indicate that alpha 1 receptors do not precondition through an adenosine intermediate but that alpha 1-adrenergic and adenosine receptors activate parallel pathways within the myocyte that can trigger and mediate protection.

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

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

Background: The vulnerability of the myocardium of a diabetic animal to an ischemic insult is controversial. To address this issue, streptozotocin-induced non-insulin-dependent diabetes (NIDD) was induced in rats, and the effects of regional myocardial ischemia were assessed by measuring infarct size., Methods and Results: Open-chest rats with NIDD and age-matched control rats underwent 30 or 45 minutes of regional ischemia and 2-hour reperfusion. Infarct size was measured by tetrazolium. Control rats had 32.0 +/- 3.3% infarction of the risk zone after a 30-minute coronary occlusion, whereas NIDD rats had significantly smaller infarcts (11.5 +/- 3.1% of the risk area, P < .005). When ischemic time was extended to 45 minutes, infarct size in control animals averaged 57.9 +/- 6.2%, whereas only 37.3 +/- 5.6% of ischemic myocardium was infarcted in NIDD rats (P < .05). In a subset NIDD group, rats experienced a period of ischemic preconditioning (three cycles of 5-minute ischemia/5-minute reperfusion) before 45-minute ischemia. Infarct size in these rats averaged only 6.9 +/- 3.0% (P < .01 vs nonpreconditioned NIDD rats with 45-minute coronary occlusions). Collateral flow was measured in NIDD rat hearts with radioactive microspheres. Collateral flow was < 1% of normal myocardial blood flow., Conclusions: We conclude that NIDD protects the heart from infarction and that this protection is not related to the development of coronary collaterals. Furthermore, preconditioning can further protect the NIDD heart.

Background: A brief coronary occlusion before a more prolonged occlusion results in less myocardial infarction than the longer occlusion alone. However, the effects of this preconditioning on recovery of systolic function after coronary occlusion have not been determined., Methods and Results: Ultrasonic crystals implanted in rabbit myocardium measured segment length in the distribution of a branch of the left coronary artery that was fitted with a snare occluder. Rabbits were randomly allocated to either nonpreconditioned or preconditioned groups. Rabbits in the latter group underwent preconditioning with a 5-minute coronary occlusion followed by 10 minutes of reperfusion. Then the coronary artery was occluded for 20 minutes in all rabbits, after which it was allowed to reperfuse for 90 minutes. The hearts were then excised, and infarct size was measured by staining with triphenyltetrazolium chloride. During coronary occlusion, all hearts except one demonstrated either akinesis or paradoxical bulging. Five minutes after release of the 20-minute occlusion, active shortening had returned in the preconditioned rabbits and averaged 27.9 +/- 16.6% of baseline shortening. At the same time, paradoxical lengthening persisted in nonpreconditioned rabbits (-15.5 +/- 19.8% of baseline). By the end of the 90-minute reperfusion period, segment shortening averaged 40.1 +/- 8.4% of baseline in preconditioned rabbits and only 6.2 +/- 12.0% in nonpreconditioned rabbits (p less than 0.05). Infarct size as a percentage of risk area was significantly smaller in preconditioned rabbits as well (3.0 +/- 1.6% versus 28.8 +/- 7.0%, p less than 0.002) and likely accounted for the improved shortening., Conclusions: We conclude that a brief coronary occlusion before a more prolonged occlusion results in not only reduced infarct size but also significantly better recovery of systolic function.

Background: Although specific guidelines for the treatment of hypercholesterolemia have been published, it is not known whether physicians treating patients likely to have lipid disorders have adopted the recommendations., Methods and Results: The approach of cardiologists to the treatment of hypercholesterolemia in a metropolitan teaching hospital was assessed by interviewing patients with chest pain who were admitted for coronary angiography in 1988-1989 and by measuring fasting blood lipid profiles. At 1 month and again 12-24 months later, patients were contacted by telephone to determine if there had been any changes in treatment. Of 95 patients evaluated, 81 had coronary artery disease. Only 17% of those with high levels of total cholesterol and/or low density lipoprotein cholesterol were being actively treated with diet and/or drugs. In the remaining patients, either lipid studies had not been done or abnormal results had not been addressed. There was little change in treatment approach during the month after the diagnostic procedure. Furthermore, the experience was similar in those patients subjected to coronary revascularization. One to 2 years after the initial intervention, 69 of the original study group could be contacted again. Although active dietary or pharmacological therapy was initiated in some individuals during this interval, it was stopped in others. Thirty-five percent of hypercholesterolemic patients were receiving targeted therapy., Conclusions: Thus, only a small proportion of patients with documented coronary artery disease and hypercholesterolemia were being actively treated for their lipid disorder, suggesting that the published treatment guidelines have not yet been fully accepted. However, an encouraging improvement in frequency of treatment of hypercholesterolemia was documented during the 1-2-year observation period.

To assess regional contractility in idiopathic hypertrophic subaortic stenosis (IHSS), a primary myopathic disorder with documented hyperdynamic ventricular contractions, systolic wall thickening and velocity of contraction of the septum and left ventricular posterior wall were measured in echocardiograms from 16 patients with IHSS and 16 normal subjects. The average thickening of the normal septum and posterior wall was 75.9+/-8.8% and 84.8+/-6.3%, respectively. The posterior wall in IHSS thickened by 75.1+/-6.8%. None of these values differed significantly. However, the increase in thickness of the IHSS septum averaged 22.5+/-2.4%, significantly less than that of either the IHSS posterior wall or the normal septum. Velocity measurements confirmed the impression of diminished septal function. The mean velocity of normal septal contraction averaged 37.0+/-2.3 mm/sec, normal posterior wall 42.3+/-2.0 mm/sec and IHSS posterior wall 55.7+/-3.5 mm/sec, whereas the septum in IHSS contracted at the rate of 26.0+/-2.5 mm/sec. Thus, the IHSS septum contracted significantly more slowly than the normal septum or IHSS posterior wall. However, the posterior wall velocity in IHSS was significantly more rapid than that measured in normal ventricles--perhaps to compensate for the septum. Normalization of all velocities for left ventricular end-diastolic internal diameter did not alter the sifnificance of the results. Consideration of IHSS as an asymmetric myopathy based on prior observations of predominantly septal hypertrophy and distorted septal cellular architecture is now supported by the above evidence of functional left ventricular asymmetry. Although the total left ventricular function in IHSS may be hyperdynamic, regional function is not uniform. The septum appears to be hypodynamic, while the contractile capacity of the posterior wall is increased.

Obstructive lesions of the main left coronary artery (LCA) were demonstrated angiographically in 73 patients, comprising 4.3% of the total population referred to us for diagnostic evaluation of chest pain. Although there were no specific historical or clinical features which could absolutely distinguish this subgroup from the larger population of coronary artery disease patients, 81% (34/42) of the double Master's exercise tests, in which the patient achieved a heart rate of at least 110 beats/min. demonstrated greater than or equal 2 mm R-ST segmental depression. Of the total group of 73, 32 were evaluated during the six-year period from 1964 to 1971 and a preliminary report made in 1972. The diagnosis in the remaining 41 patients was established in the 1/2 year period from 1971 to 1973. The initial 32 patients were seen before the significance of a main LCA lesion was appreciated. In this subgroup there were five deaths at the time of cardiac catheterization. However, in the more recent group improved recognition of patients with possible main LCA disease prior to catheterization has led to a much lower death rate related to diagnostic catheterization. Only one of the last 41 patients undergoing coronary angiography has died. Nineteen patients were managed medically. Of this group 17 were considered to be under the same risk of death as the surgical candidates. Their mortality rate, as high. The risk of hying was 43.6% after 24 months, 51.1% after 36 months, and 73.6% after 42 months of observation. Although the initial surgical experience, using internal mammary artery implants and saphenous venin bypass grafts, was associated with a high mortality, direct revascularization surgery over the last 2 1/2 years has been accomplished with a perioperative mortality of only 6.2%. All deaths in the group of 40 patients receiving elective revasularization with saphenous vein bypass grafts occurred in the first six months following surgery. The risk of dying was 12.5% after six months of observation and was unchanged for the remainder of the follow-up period. After 21 months the difference in survival between the two groups is statistically significant (P less than 0.05). The one late death among the surgical survivors occurred four months after the operative procedure and was related to noncardiovaxcular surgical complications. The surgical survivors have been followed for an average of 27 months. Thus revascularization surgery has improved the prognosis for patients with main LCA disease. We currently advise prompt evaluation for any patient suspected of having this type of obstruction and urgent idrect revascularization surgery if this lesion is demonstrated angiographically.

Pulmonary arterial early diastolic waves (V waves) were investigated in patients and experimental animals with mitral regurgitation. V waves exceeding systolic pressure in the pulmonary artery were recorded in the main pulmonary artery with micromanometer catheters both in patients and animals, eliminating the possibility of catheter artifact. In experimental animals, aortic closure preceded pulmonic closure by 33 +/- 12 msec at baseline. With the creation of acute mitral insufficiency, a pulmonary arterial V wave occurred in six of eight animals. Early pulmonic valve closure occurred only in the six animals with a pulmonary arterial V wave. In these animals, pulmonic closure preceded aortic closure by 28 +/- 7 msec during mitral insufficiency (p less than .05). Of 70 patients with severe mitral regurgitation at cardiac catheterization, 14 had a pulmonary arterial V wave. In five patients recordings with micromanometer catheters were made and early pulmonic closure was also observed in four of these patients who had pulmonary arterial V waves at rest or upon provocation. Patients with pulmonary arterial V waves had a more acute onset of symptoms, shorter duration of mitral regurgitation, higher pulmonary capillary wedge V waves, and lower pulmonary arterial resistances than patients without them and were more likely to have nonrheumatic mitral regurgitation.

To examine the effects of coronary collateral development on thallium-201 (201Tl) distribution the left circumflex coronary artery was ligated in eight dogs. Three days later these animals ran on a treadmill, and 201-thallous chloride was injected into the right atrium at peak exercise. Scintigraphic scanning was begun within 10 min and continued for 3 hr. Scanning was repeated weekly for 6 weeks. In the last week radioactive microspheres were injected into the left atrium at peak exercise to measure regional myocardial blood flow. The scintigraphically determined disparity between perfusion of the ischemic and normal myocardium was most marked at 3 days after ligation. This difference gradually lessened over the first 4 weeks until there was no difference in 201Tl distribution to normally perfused myocardium and tissue distal to the ligation. Concomitant with the improvement in the scintigrams, exercise hemodynamics also improved over this 4 week period with significant increases in cardiac output and decreases in left atrial pressure. Serial coronary angiographic studies in two animals demonstrated the appearance of collaterals in the initial weeks after coronary occlusion, and by 4 weeks the left circumflex artery distal to the obstruction was completely opacified by collateral flow. The ratio of directly measured exercise blood flow to the left circumflex and normally perfused tissues was 0.89 +/- 0.08 at 6 weeks after ligation. Scintigraphic 201Tl redistribution after 3 hr also changed over the weeks after ligation. Three days after ligation washout from the ischemic area was significantly slower than that from the normal myocardium. By 6 weeks loss of 201Tl from the two regions occurred at nearly equal rates. Thus myocardial perfusion and function during exercise after coronary occlusion are dynamic events that change with time. It is likely that coronary collateral development is responsible for these phenomena. Therefore coronary collaterals do have salutary effects in the dog.

To examine the effects of acute myocardial infarction on mitral valve diastolic velocity, echocardiograms were performed in 18 patients on admission, daily in the Coronary Care Unit, and at 3-day intervals during the remainder of hospitalization. These patients were divided into three groups based on the time interval between onset of symptoms and initial echocardiogram. Five of six patients admitted within 5 hours of onset of myocardial infarction had a triphasic response of mitral valve diastolic velocity with a transient rise above initial values, followed by a fall to below initial values, and then a slow rise during recovery. Seven of eight patients admitted 1-2 days after onset of myocardial infarction had a biphasic response, i.e., a fall from initial values and then a slow rise. Four patients admitted later in the course of myocardial infarction had a monophasic response, i.e. low initial velocity followed by a slow recovery. We conclude that in patients with myocardial infarction the mitral valve diastolic velocity following myocardial infarction shows a triphasic response which may appear biphasic or monophasic depending on the interval between myocardial infarction and admission. The temporal pattern of mitral valve diastolic velocity changes may reflect the dynamic alterations of myocardial function and compliance that are occurring after acute myocardial infarction and during the recovery period.

To determine if oxygen free radical scavengers administered before coronary artery reperfusion can limit reperfusion arrhythmias, increase the return of regional function in ischemic myocardium, and reduce tissue necrosis at 1 week after 90-minute coronary artery occlusion and reperfusion, conscious dogs were treated with superoxide dismutase (SOD) and catalase before and for 1 hour after coronary artery reperfusion. Another group was treated with recombinant SOD (rSOD) because the commercially available SOD and catalase contained endotoxin. The conscious dogs were studied 3-4 weeks after implanting left ventricular pressure gauges, ultrasonic wall thickness gauges in the posterior left ventricular wall, left atrial catheters, and arterial catheters, Doppler flow transducers, and hydraulic occluders on the left circumflex coronary artery. The only beneficial effect observed was that the number of arrhythmic beats per minute in the rSOD-treated group was significantly lower (p less than 0.05) when compared with a control group after coronary artery reperfusion. Treatment neither increased the amount of recovery of wall thickening in the ischemic zone nor reduced infarct size when expressed either as a percentage of the area at risk or as a function of collateral blood flow in the ischemic zone. For example, infarct size as a percentage of the area at risk was 32.6 +/- 5.8%, 37.4 +/- 6.4%, 28.3 +/- 5.1% in the control, SOD and catalase-, and rSOD-treated groups, respectively. Thus, although treatment with oxygen free radical scavengers invoked a transient reduction in the number of reperfusion arrhythmias, this treatment in conscious dogs failed to improve regional myocardial dysfunction or reduce the amount of necrosis when compared with a control group. The lack of a sustained salutary effect may indicate that longer periods of treatment with free radical scavengers are required in chronic preparations.

12 patients who had atrial flutter without clinical, echocardiographic or angiographic evidence of aortic insufficiency were studied with simultaneous echo- and phonocardiograms. In patients with high-grade atrioventricular (AV) block, the mitral valve opened and closed with each flutter wave. Of seven patients, two had persistent and five had intermittent early mitral valve closure before QRS inscription. In five patients (three with 2:1 AV block) the mitral valve closed on time. In one patient with a mitral valve prosthesis, echocardiography and cinefluorography demonstrated closure during mid-diastole, with reopening in late diastole after a flutter wave. Final valve closure occurred before the onset of the QRS, and each closure was associated with a click. Simultaneous phonocardiographic analysis in these patients demonstrated that the first heart sound intensity was inversely related to the degree of mitral valve preclosure. This relationship was independent of the length of the RR interval. Thus, atrial flutter independent of any other cause of abnormal hemodynamics may produce early mitral valve closure. The echocardiographic finding of premature mitral arrhythmias, may not have the same diagnostic or prognostic significance previously described in patients with sinus rhythm and normal AV conduction.

To determine the causes of cardiac failure during cardiac tamponade in man, we studied left ventricular volume and function in eight patients during pericardiocentesis using gated equilibrium radionuclide ventriculography. In the seven patients with clinical and hemodynamic evidence of cardiac tamponade, end-diastolic and end-systolic volumes increased progressively as the initial 500 ml of fluid were removed; the most marked increase occurred during the removal of the first 200 ml of pericardial fluid. After removal of 500 ml of pericardial fluid, end-diastolic volume increased from 52 +/- 8 ml to 111 +/- 13 ml (p less than 0.05) and end-systolic volume from 17 +/- 5 ml to 34 +/- 7 ml (p less than 0.05). Additional aspiration of fluid resulted in no further changes in left ventricular volume. The ejection fraction averaged 70% before removal of fluid and was unchanged by pericardiocentesis. In the one patient who did not have hemodynamic evidence of tamponade, there were only minor changes in left ventricular volumes and ejection fraction. These data suggest that pump function of the left ventricle is well preserved in cardiac tamponade, and that the diminution in stroke volume and consequent cardiovascular collapse seen in tamponade are due to marked underfilling of the ventricle.