The blood contribution to early myocardial reperfusion injury is amplified in diabetes

Cardiovascular disease is excessive in diabetes, and blood cell function is altered. It is not clear, however, if alterations in the blood contribute to the excessive cardiovascular complications of this disease. In this study, we compared the contribution of nondiabetic and diabetic blood to myocardial reperfusion injury. The recovery of cardiac contractile function following no-flow ischemia was studied in isolated diabetic and nondiabetic rat hearts perfused with diabetic or nondiabetic diluted whole blood. Hearts were isolated from 10- to 12-week-old diabetic (streptozotocin, 65 mg/kg, i.v.) and nondiabetic rats and perfused with a Krebs-albumin-red cell solution (K2RBC, Hct 20%). After a 30-min pre-ischemic control period, during which cardiac pump function was evaluated, diabetic and nondiabetic hearts were perfused for 5 min with diluted whole blood (DWB; Hct 20%) collected from either diabetic or nondiabetic donor animals. Coronary flow was then stopped and the hearts subjected to 30 min of no-flow ischemia. Following ischemia, the hearts were reperfused with the K2RBC perfusate. Cardiac contractile function was evaluated throughout the 60-min reperfusion period. Six groups were studied: diabetic and nondiabetic hearts perfused before ischemia with either K2RBC, nondiabetic DWB (NDDWB), or diabetic DWB (DDWB). Perfusion with DWB prior to ischemia impaired the recovery of contractile function in all cases. The impairment to recovery was greater with DDWB than with NDDWB. Although diabetic hearts perfused with K2RBC throughout recovered quite well, the effect of DDWB perfusion in the diabetic hearts was dramatic. In an effort to determine why diabetic blood impaired functional recovery, measures of blood filterability and the generation of reactive oxygen species (ROS) were made. We found that diabetic blood was less filterable than nondiabetic blood; that is, the diabetic blood cells tended to plug the 5-microm filter pores more readily than the nondiabetic blood cells. Also, we found that the diabetic blood was capable of generating significantly greater ROS (oxygen free radicals) than nondiabetic blood (P < 0.05). These findings suggest that the blood contribution to myocardial reperfusion injury is amplified in diabetes. A tendency for diabetic blood cells to plug capillary-sized pores and show enhanced oxygen free radical production may account for the excessive contribution of diabetic blood to reperfusion injury in the heart.     

Use of immunoblot assay to define serum antibody patterns associated with Helicobacter pylori infection and with H. pylori-related ulcers

OBJECTIVE: We evaluated the effect of pretreatment with nitric oxide precursor before ischemia on recovery with reperfusion in rat hearts. METHODS: Isolated rat hearts were perfused with Krebs-Henseleit buffer without (C group) or with 3 mmol/L L-arginine (A group) before 30 minutes of ischemia. The left ventricular function, including heart rate, developed pressure, maximal dp/dt, and coronary flow, were measured before pretreatment and after 10 and 30 minutes of reperfusion. Cyclic guanosine monophosphate (by radioimmunoassay), calcium (by absorption spectrophotometry), and inositol 1,4,5-triphosphate synthesized from tritiated myo-inositol (by ion-exchange chromatography preceding counting) were measured at the same times and immediately after ischemia. RESULTS: Recovery of ventricular function was significantly greater in the A group than in the C group. Pretreatment increased postischemic cyclic guanosine monophosphate content compared with the preischemic level (from 1.06 +/- 0.12 to 1.94 +/- 0.09 pmol/mg protein, p < 0.05). No change in cyclic guanosine monophosphate was evident in the C group. In the C group, inositol triphosphate content increased after 10 minutes of reperfusion beyond the preischemic level (from 0.53 +/- 0.023 to 1.15 +/- 0.045 cpm x 10(-3)/gm, p < 0.05) as did calcium at 30 minutes (from 4.12 +/- 0.164 to 6.86 +/- 0.544 mmol/gm dry weight). In the A group, both of these increases were significantly attenuated. CONCLUSION: These data suggest that L-arginine pretreatment may reduce calcium overload by increasing cyclic guanosine monophosphate production, which in turn downregulates inositol triphosphate synthesis during reperfusion.     

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

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

Increase in incidence of insulin-dependent diabetes mellitus among children in Finland

It has been proposed that NO may function as an endogenous cardioprotectant. We have investigated whether modulation of NO levels (detected in coronary effluent by chemiluminescence) by a blocker of its synthesis, by supplementation of its precursor, and by administration of an NO donor can influence reperfusion arrhythmias in the isolated rat heart. Rat hearts were perfused with modified Krebs' solution and subjected to 5, 35, or 60 minutes of left regional ischemia followed by 10 minutes of reperfusion. NG-Nitro-L-arginine methyl ester (L-NAME), which blocks NO synthase, increased the incidence of reperfusion-induced ventricular fibrillation (VF) from 5% in the control condition to 35% after 60 minutes of ischemia (n = 20, P < .05). The profibrillatory effect of L-NAME was prevented in hearts coperfused with 1 or 10 mmol/L L-arginine (an NO precursor) but persisted in hearts coperfused with D-arginine (1 mmol/L). L-NAME did not increase VF susceptibility in hearts reperfused after 5 or 35 minutes of ischemia. L-NAME caused sinus bradycardia (264 +/- 10 versus 309 +/- 5 bpm in control groups, P < .05) and reduced coronary flow before ischemia (6.2 +/- 0.6 versus 9.2 +/- 0.6 mL.min-1.g-1 tissue in controls, P < .05). L-NAME reduced coronary effluent NO levels after 60 minutes of ischemia; during the first minute of reperfusion, values were reduced from 1457 +/- 422 to 812 +/- 228 pmol.min-1.g-1 (P < .05). This effect was prevented by coperfusion with L-arginine (10,344 +/- 1730 pmol.min-1.g-1, P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

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.     

Profound underestimation of glucose uptake by [18F]2-deoxy-2-fluoroglucose in reperfused rat heart muscle

BACKGROUND: [18F]2-deoxy-2-fluoroglucose (FDG) is widely used as a tracer for glucose uptake in ischemic heart muscle. We tested the effects of low-flow ischemia and reperfusion on the ratio of tracer/tracee (lumped constant, LC). METHODS AND RESULTS: Isolated working rat hearts were perfused with Krebs-Henseleit buffer containing only glucose 5 mmol/L (group 1) or glucose 5 mmol/L plus oleate 0.4 mmol/L (group 2, fed; group 3, fasted). Dynamic glucose uptake was measured simultaneously with [2-3H]glucose and with FDG. After 20 minutes, coronary flow was reduced by 75% for 30 minutes before it was returned to control conditions for the final 20 minutes. Hexokinase activity in the cytosolic and mitochondrial fractions and tissue metabolites were determined. Rates of glucose uptake were highest when glucose was the only substrate. Glucose uptake, FDG uptake, and the LC increased during ischemia only in group 3. There was no change of these parameters during ischemia in groups 1 and 2. FDG uptake decreased significantly with reperfusion in groups 2 and 3, and there was a striking fall in the LC (from >1.0 to <0.2, P<.001). The fall in the LC was associated with a significant increase in intracellular free glucose. Neither ischemia nor reperfusion affected the kinetic properties of hexokinase. CONCLUSIONS: FDG profoundly underestimates glucose uptake during reperfusion in the presence of fatty acids. In the fasted state, however, FDG overestimates glucose uptake during ischemia. The results indicate limitations in the use of FDG to quantify myocardial glucose uptake in human heart.     

Alterations in pulmonary surfactant composition and activity after experimental lung transplantation

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

Synergism between platelets and neutrophils in provoking cardiac dysfunction after ischemia and reperfusion: role of selectins

BACKGROUND: Neutrophils (PMNs) are known to contribute to both cardiac dysfunction and myocardial necrosis after reperfusion of an ischemic heart. Moreover, platelets are also important blood cells that can aggravate myocardial ischemic injury. This study was designed to test the effects of PMNs and platelets separately and together in provoking cardiac dysfunction in isolated perfused rat hearts after ischemia and reperfusion. METHODS AND RESULTS: Control rat hearts not subjected to ischemia were perfused without blood cells for 80 minutes. Additional control rat hearts were perfused with 75x106 PMNs, with 100x106 platelets, or with 75x106 PMNs+100x106 platelets over a 5-minute perfusion followed by a 75-minute observation period. No significant reduction in coronary flow, left ventricular developed pressure (LVDP), or the first derivative of LVDP (dP/dtmax) was observed at the end of the observation period in any nonischemic group. Similarly, global ischemia (I) for 20 minutes followed by 45 minutes of reperfusion (R) produced no sustained effects on the final recovery of any of these parameters in any group of hearts perfused in the absence of blood cells. However, I/R hearts perfused with either PMNs or platelets alone exhibited decreases in these variables of 10% to 12% (P<0.05 from control). Furthermore, I/R hearts perfused with both PMNs and platelets exhibited decreases of 50% to 60% in all measurements of cardiac function (P<0.001). These dual-cell-perfused I/R hearts also exhibited marked increases in cardiac myeloperoxidase (MPO) activity, indicating a significant PMN infiltration, and enhanced P-selectin expression on the coronary microvascular endothelium. All cardiodynamic effects as well as MPO accumulation and PMN infiltration were markedly attenuated by a sialyl LewisX-oligosaccharide or a recombinant soluble P-selectin ligand, which inhibits selectin-mediated cell adhesion. CONCLUSIONS: These results provide evidence that platelets and neutrophils act synergistically in provoking postreperfusion cardiac dysfunction and that this may be largely due to cell-to-cell interactions mediated by P-selectin. These findings may help explain the reperfusion injury phenomenon.     

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.     

Clearance of technetium 99m N-NOET in normal, ischemic-reperfused, and membrane-disrupted myocardium

BACKGROUND: Technetium 99m-labeled bis(N-ethoxy, N-ethyl dithiocarbamato) nitrido technetium(v) (99mTcN-NOET) is a new neutral cardiac perfusion imaging agent that has been shown to have very high uptake and retention in vitro. The purpose of this study was to determine the clearance kinetics of 99mTcN-NOET in control, ischemic-reperfused, and membrane-disrupted myocardium. METHODS AND RESULTS: After a 100 microCi (3.7 x 10(6) Bq) bolus of 99mTcN-NOET was injected, myocardial clearance was monitored for 1 hour by the use of a sodium iodide detector in 30 isolated, Krebs-Henseleit (KH) perfused rat hearts. Seven hearts were used as controls (group 1). In seven ischemic-reperfused hearts, tracer administration and uptake was followed by 30 minutes of no flow and 1 hour of reflow (group 2). In six additional ischemic-reperfused hearts, tracer administration was followed by deprivation of flow for 1 hour followed by 1 hour of reflow (group 3). Six hearts were perfused with a 0.5% Triton X-100 KH perfusate for 1 hour (group 4). Four hearts were perfused with KH for 10 minutes, followed by cyanide for 10 minutes (group 5). This cycle was repeated three times. Activities remaining in each heart at the end of each experiment were quantitated, and activity at peak uptake was calculated. The 99mTcN-NOET myocardial clearance was near linear in the control (0.6 +/- 0.4) and both ischemic-reperfused groups with virtually no fractional clearance (1.2% +/- 0.6% and 2.1% +/- 0.6%, respectively; p = NS). In the Triton X-100 membrane-disrupted hearts, clearance was substantial (94.2% +/- 4.0%; p < 0.0001 compared with the control and ischemic-reperfused groups). Cyanide treatment produced rapid clearance, which was arrested by a return to the standard KH perfusate. Peak uptake as a percentage of injected dose was 74.9% +/- 1.4% for all groups combined. CONCLUSION: Thus 99mTcN-NOET has extremely high myocardial retention after 1 hour in normal myocardium and is not significantly affected by ongoing myocardial ischemia or reperfusion injury in this model. Clearance is increased markedly in extreme conditions of membrane disruption. These data are consistent with the concept that 99mTc-NOET is localized predominantly in or on cell membranes. 99mTcN-NOET is a promising, new myocardial perfusion imaging agent that exhibits a stable myocardial distribution in the setting of acute developing injury.     

Release of 4-hydroxynonenal, an aldehydic mediator of inflammation, during postischaemic reperfusion of the myocardium

For the first time it was demonstrated that 4-hydroxynonenal (HNE) is formed by the myocardium. 1 to 2 pmol HNE/min/mg protein were released from isolated perfused hearts of 18-month-old WKY rats during a normoxic period of perfusion. During the first minutes of reperfusion following 30 min of ischaemia, the mean value of HNE release increased in comparison to pre-ischaemic HNE release (pre-ischaemic control). However, the alterations were significant only in the second minute of reperfusion. HNE liberation significantly intensified during the early reperfusion period of hearts of 18-month-old spontaneously hypertensive rats (SHR, with cardiac hypertrophy and congestive heart failure) in comparison with the pre-ischaemic control period. Furthermore, HNE liberation from those hearts was higher than from hearts of normotensive control animals (WKY rats). Maximum quantities were observed 2 min after ischaemia, with 6 to 10 pmol HNE/min/mg protein. The results suggest that the formation of chemotactic products of radical-induced lipid peroxidation, such as HNE, is markedly increased in reperfused hypertrophic and failing myocardium, and emphasize the role of HNE as a possible chemotactic agent during postischaemic reoxygenation.     

Neural dysfunction and metabolic imbalances in diabetic rats. Prevention by acetyl-L-carnitine

The rationale for these experiments is that administration of L-carnitine and/or short-chain acylcarnitines attenuates myocardial dysfunction 1) in hearts from diabetic animals (in which L-carnitine levels are decreased); 2) induced by ischemia-reperfusion in hearts from nondiabetic animals; and 3) in nondiabetic humans with ischemic heart disease. The objective of these studies was to investigate whether imbalances in carnitine metabolism play a role in the pathogenesis of diabetic peripheral neuropathy. The major findings in rats with streptozotocin-induced diabetes of 4-6 weeks duration were that 24-h urinary carnitine excretion was increased approximately twofold and L-carnitine levels were decreased in plasma (46%) and sciatic nerve endoneurium (31%). These changes in carnitine levels/excretion were associated with decreased caudal nerve conduction velocity (10-15%) and sciatic nerve changes in Na(+)-K(+)-ATPase activity (decreased 50%), Mg(2+)-ATPase (decreased 65%), 1,2-diacyl-sn-glycerol (DAG) (decreased 40%), vascular albumin permeation (increased 60%), and blood flow (increased 65%). Treatment with acetyl-L-carnitine normalized plasma and endoneurial L-carnitine levels and prevented all of these metabolic and functional changes except the increased blood flow, which was unaffected, and the reduction in DAG, which decreased another 40%. In conclusion, these observations 1) demonstrate a link between imbalances in carnitine metabolism and several metabolic and functional abnormalities associated with diabetic polyneuropathy and 2) indicate that decreased sciatic nerve endoneurial ATPase activity (ouabain-sensitive and insensitive) in this model of diabetes is associated with decreased DAG.     

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

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

Incremental iron overload during reperfusion progressively augments oxidative injury

OBJECTIVE: To determine if a relationship exists between the extent of iron-catalyzed injury and the degree of tissue iron overload during reperfusion. METHODS: To selectively increase tissue iron only during early reperfusion, isolated, buffer perfused rabbit hearts were exposed to 20 microM Fe(2+)-100 microM ADP during the last 3 minutes of ischemia and the initial 4 minutes of reperfusion. Control groups were exposed to ADP and iron-ADP regimens that did not increase intracellular iron. All the hearts received 30 minutes of normothermic global ischemia and 30 minutes of reperfusion. Heart function was monitored continuously throughout each experiment. Tissue iron and biochemical markers were analyzed at the end of experiments. RESULTS: Hemodynamic recovery was decreased and tissue lipid peroxide levels were increased in the 20 microM Fe(2+)-100 microM ADP group compared to controls. The recoveries of developed pressure and positive/negative dP/dT at 30 minutes of reperfusion were negatively correlated with tissue iron levels, while cytosol and membrane lipid peroxide levels correlated positively with the iron levels during reperfusion. CONCLUSION: The extent of oxidative injury during reperfusion was directly related to the tissue iron burden present during reperfusion. Increased lipid peroxidation was the principal chemical marker of iron-catalyzed injury.     

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.     

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.     

HPTLC analysis of sphingomylein, ceramide and sphingosine in ischemic/reperfused rat heart

Since the sphingomylein-ceramide-sphingosine pathway, especially ceramide, has been shown to induce programmed cell death (apoptosis), and since apoptosis may be involved with ischemic/reperfused injury in the heart, it became desirable to quantitate the three components in ischemic/reperfused rat heart. One group of rat hearts (n = 6) was isolated and perfused with Krebs-Henseleit buffer using the Langendorff non-recirculating mode. The hearts were perfused for 10 min, made ischemic for 30 min and reperfused for 120 min. Hearts were collected and stored at - 70 degrees C before ischemia, after ischemia and after 30, 60 and 120 min of reperfusion. The hearts were homogenized, and lipids were extracted using the Folch method. The lipids were then chromatographed on Whatman silica gel 60 A high-performance thin-layered chromatography (HPTLC) plates. The plates were developed with iodine, photographed using Photoshop software and quantitated using NIH Imaging software. The results show a 50% decrease of sphingomylein during reperfusion with a corresponding increase in ceramide with sphingosine showing a smaller decrease as compared with the ceramide increase.     

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.     

Salient factors that influence the meaning of family caregiving for frail elderly parents in Japan from a historical perspective

Cardiac reperfusion and aging are associated with increased rates of mitochondrial free radical production. Mitochondria are therefore a likely site of reperfusion-induced oxidative damage, the severity of which may increase with age. 4-Hydroxy-2-nonenal (HNE), a major product of lipid peroxidation, increases in concentration upon reperfusion of ischemic cardiac tissue, can react with and inactivate enzymes, and inhibits mitochondrial respiration in vitro. HNE modification of mitochondrial protein(s) might, therefore, be expected to occur during reperfusion and result in loss in mitochondrial function. In addition, this process may be more prevalent in aged animals. To begin to test this hypothesis, hearts from 8- and 24-month-old rats were perfused in Langendorff fashion and subjected to periods of ischemia and/or reperfusion. The rate of state 3 respiration of mitochondria isolated from hearts exposed to ischemia (25 min) was approximately 25% less than that of controls, independent of age. Reperfusion (40 min) caused a further decline in the rate of state 3 respiration in hearts isolated from 24- but not 8-month-old rats. Furthermore, HNE modification of mitochondrial protein (approximately 30 and 44 kDa) occurred only during reperfusion of hearts from 24-month-old rats. Thus, HNE-modified protein was present in only those mitochondria exhibiting reperfusion-induced declines in function. These studies therefore identify mitochondria as a subcellular target of reperfusion damage and a site of age-related increases in susceptibility to injury.