Faculty Bibliography

Lipopolysaccharide (LPS) produces varied systemic metabolic effects. We studied the effects of LPS on the cardiac fatty acid profile and its relationship to energy metabolism and inflammatory mediators that included TNF-alpha and nitric oxide synthase (NOS) in 10-day-old neonatal rat pups. Rat pups received an i.p. injection of LPS after a 4-hour starvation period, followed by collection of blood and cardiac tissue 4 h following LPS administration. Compared to controls, LPS induced significant hypoglycemia and hyperlactacidemia, suggesting the development of endotoxic shock. The result was a significant depression in total fatty acid levels as well as non-esterified fatty acid in the cardiac tissue of the LPS-treated pups. In addition, LPS-treated pups also showed a significant increase in TNF-alpha, NOS levels with a depressed redox state and energy metabolism in cardiac tissue. These observations suggest that endotoxic shock in 10-day-old rat pups induces a systemic inflammatory response with a depression in fatty acid metabolism that may contribute to myocardial failure

Fatty acid oxidation defects are being increasingly identified as causes of abnormal heart function and sudden death in children. Children with medium-chain acyl-coenzyme A (acyl-CoA) dehydrogenase defects can metabolize fatty acids labeled in the carboxylic acid end of the compound. Accordingly, our goal was to label a long-chain fatty acid in the omega-position and evaluate its myocardial kinetics. METHODS: Heptadecanoic acid, a 17-carbon fatty acid, was labeled in the C-17 position with (11)C by the general process of coupling (11)C-methyliodide to t-butyl-15-hexadecanoate. Yield was approximately 5%-10% end-of-bombardment. Subsequently, evaluation studies were performed on isolated perfused rat hearts and in intact, anesthetized dogs. The myocardial uptake and efflux of 17-(11)C-heptadecanoic acid were compared with those of 1-(11)C-palmitate. RESULTS: With the exception of delayed efflux of tracer reflecting the temporal delay for beta-oxidation, the washout of 17-(11)C-heptadecanoic acid from the heart mirrored that of 1-(11)C-palmitate in isolated rat hearts and in intact dogs with PET. CONCLUSION: 17-(11)C-Heptadecanoic acid may be a useful tracer for the identification of defects in fatty acid metabolism in subjects with medium- and short-chain fatty acid oxidation defects

BACKGROUND: A number of experimental studies have shown that increasing glucose use or decreasing accumulation of long-chain acyl carnitines (LCAC) protect ischemic hearts. METHODS: To evaluate the relative importance of these two strategies in protecting ischemic myocardium, isolated rat hearts (n = 6 in each group) were paced at 300 bpm and subjected to 50 min of low-flow ischemia followed by 60 min of reperfusion. Buffer contained 0.4 m mol/l albumin, 0.4 m mol/l palmitate, and 70 mU/l insulin, and either normal glucose (5 m mol/l) (CON), high glucose (10 m mol/l total) (HG, known to increase glucose use), 5 m mol/l glucose and niacin (10 micromol/l) (NIA, known to increase glucose use and decrease LCAC) or carnitine (10 m mol/l) (CAR, known to increase glucose use and decrease LCAC). Separate groups of hearts were perfused in the presence of 10 micromol/l cytochalasin-B (CB), an inhibitor of insulin-sensitive glucose transporters. RESULTS: Ischemic injury, as assessed by creatine kinase (CK) release was diminished by an average of 50% in HG, NIA, and CAR hearts, and the percentage recovery of left ventricular (LV) function with reperfusion was enhanced by approximately 20% compared with CON hearts (P < 0.05 for each comparison). Cytochalasin-B abolished all of the salutary effects. Long-chain acyl carnitines levels were higher in HG hearts compared with NIA- and CAR-treated hearts ( P < 0.05), but ischemic protection and functional recovery was greater in HG hearts. CONCLUSIONS: The data support the adjunctive use of agents that promote glucose uptake during ischemia and suggest that increasing glucose use is more important than decreasing LCAC in the protection against ischemic injury or in the recovery of contractile function

Endothelial CD39 metabolizes ADP released from activated platelets. Recombinant soluble human CD39 (solCD39) potently inhibited ex vivo platelet aggregation in response to ADP and reduced cerebral infarct volumes in mice following transient middle cerebral artery occlusion, even when given 3 hours after stroke. Postischemic platelet and fibrin deposition were decreased and perfusion increased without increasing intracerebral hemorrhage. In contrast, aspirin did not increase postischemic blood flow or reduce infarction volume, but did increase intracerebral hemorrhage. Mice lacking the enzymatically active extracellular portion of the CD39 molecule were generated by replacement of exons 4-6 (apyrase-conserved regions 2-4) with a PGKneo cassette. Although CD39 mRNA 3' of the neomycin cassette insertion site was detected, brains from these mice lacked both apyrase activity and CD39 immunoreactivity. Although their baseline phenotype, hematological profiles, and bleeding times were normal, cd39(-/-) mice exhibited increased cerebral infarct volumes and reduced postischemic perfusion. solCD39 reconstituted these mice, restoring postischemic cerebral perfusion and rescuing them from cerebral injury. These data demonstrate that CD39 exerts a protective thromboregulatory function in stroke

Aldose reductase, a member of the aldo-keto reductase family, has been implicated in the development of vascular and neurological complications in diabetes. Despite recent studies from our laboratory demonstrating protection of ischemic hearts by an aldose reductase inhibitor, the presence and influence of aldose reductase in cardiac tissue remain unknown. Our goal in this study was to isolate and characterize the kinetic properties of cardiac aldose reductase, as well as to study the impact of flux via this enzyme on glucose metabolism and contractile function in hearts subjected to ischemia-reperfusion. Results demonstrate that ischemia increases myocardial aldose reductase activity and that these increases are, in part, due to activation by nitric oxide. The kinetic parameter of cardiac aldose reductase (Kcat) was significantly higher in ischemic tissues. Aldose reductase inhibition increased glycolysis and glucose oxidation. Aldose reductase inhibited hearts, when subjected to ischemia/reperfusion, exhibited less ischemic injury and was associated with lower lactate/pyruvate ratios (a measure of cytosolic NADH/NAD+), greater tissue content of adenosine triphosphate, and improved cardiac function. These findings indicate that aldose reductase is a component of ischemic injury and that pharmacological inhibitors of aldose reductase present a novel adjunctive approach for protecting ischemic hearts

INTRODUCTION: Fasting has been shown to limit ischemic injury and improve functional activity after global ischemia. Because calcium overload is considered a mechanism of ischemic injury, we hypothesized that fasting would limit the accumulation of intracellular calcium [Ca]i during ischemia, potentially due to reduced accumulation of intracellular sodium [Na]i. METHODS: To address this hypothesis, hearts isolated from rats fed either a normal diet or fasted for 24 hours underwent 20 min of global ischemia at 37 degrees. In addition to functional parameters, [Na]i and [Ca]i were measured using 21Na and 19F spectroscopy using thulium-DOTP-5 and 5F-BAPTA, respectively. In vitro measurement of sarcoplasmic reticulum calcium uptake and release, as well as activity of the sarcolemmal Na-Ca exchanger, was performed in hearts from fed and fasted animals under baseline and ischemic conditions. RESULTS: Hearts from fasted animals showed greater recovery of developed pressure (37+/-9 vs. 11+/-6 cm H2O, p < 0.05) and less contracture (end-diastolic pressure 25+/-2 vs. 47+/-2 cm H2O, p < 0.05) by the end of the reperfusion period. [Na]i was similar in the 2 groups during the first half of the ischemic period, albeit with a higher concentration of [Na]i in hearts from fed compared to fasted animals at reperfusion. Fasting markedly limited calcium accumulation during ischemia, with end-ischemic calcium being 419+/-46 nM in the hearts from fasted animals and 858+/-140 nM in the hearts from fed animals (p < 0.01). There was no significant effect of fasting on calcium uptake or release by the SR, nor on sarcolemmal Na-Ca exchange activity. CONCLUSIONS: Fasting for 24 hours improves functional recovery and markedly limits [Ca]i accumulation during ischemia and early reperfusion. The mechanism for this phenomenon remains to be elucidated

OBJECTIVE: Metabolic interventions that promote glucose use during ischemia have been shown to protect the myocardium and improve functional recovery on reperfusion. In this study we evaluated if cardioprotection can be accomplished by inhibiting fatty acid uptake, which would be expected to increase glycolytic metabolism. METHODS: Diisothiocyanostilbene sulfonic acid (DIDS), commonly used to inhibit Band-3 mediated anion exchanger, and has also been demonstrated to inhibit fatty acid transport in adipocytes, was used to inhibit fatty acid uptake prior to ischemia. Isolated rat hearts were perfused with buffer containing 5 mM glucose, 70 mU/l insulin, 0.4 mM palmitate, and 0.4 mM albumin, paced at 300 beats/min, and subjected to 50 min of low-flow ischemia followed by 60 min of reperfusion. RESULTS: Ischemic injury, as assessed by creatine kinase release, was diminished in hearts perfused with DIDS (334+/-72 in DIDS vs. 565+/-314 IU/g dry wt in controls, P<0.04). Increases in LVEDP during ischemia were attenuated (8+/-3 mmHg in DIDS vs. 15+/-18 mmHg in controls, P<0.03) and the % recovery of LV function with reperfusion was enhanced in DIDS-treated hearts (78+/-10% of baseline in DIDS vs. 62+/-19% of baseline in controls, P<0.04). These beneficial effects of DIDS were associated with increased glucose metabolism and ATP content during ischemia and reperfusion. Furthermore, treatment with DIDS lowered the accumulation of long chain acyl carnitines. CONCLUSIONS: This study demonstrates that DIDS protects ischemic myocardium, and is associated with inhibition of fatty acid uptake, improved glucose metabolism, and enhanced functional recovery on reperfusion. The data presented here suggest a potential role for therapeutic agents that lower fatty acid uptake as a metabolic adjunct in the treatment of myocardial ischemia

Diabetes increases both the incidence of cardiovascular disease and complications of myocardial infarction and heart failure. Studies using diabetic animals have shown that changes in myocardial sodium transporters result in alterations in intracellular sodium (Na(i)) homeostasis. Because the changes in sodium homeostasis can be due to increased entry of Na+ via the electroneutral Na+-K+-2Cl- cotransporter (NKCC), we conducted experiments in acute diabetic hearts to determine if 1) net inward cation flux via NKCC is increased, 2) this cotransporter contributes to a greater increase in Na(i) during ischemia, and 3) inhibition of NKCC limits injury and improves function after ischemia-reperfusion. These issues were investigated in perfused type I diabetic and nondiabetic rat hearts subjected to ischemia and 60 min of reperfusion. A group of diabetic and nondiabetic hearts was perfused with 5 microM of bumetanide, an inhibitor of NKCC. Flux via NKCC, Na(i), and ATP was measured in each group with the use of radiotracer 86Rb, 23Na, and 31P nuclear magnetic resonance spectroscopy, respectively, whereas ischemic injury was assessed by measuring creatine kinase release on reperfusion. Cation flux via NKCC, as measured by 86Rb uptake, was significantly increased in diabetic hearts. Inhibition of NKCC significantly reduced ischemic injury in diabetic hearts, improved functional recovery on reperfusion, attenuated the ischemic rise in Na(i), and conserved ATP during ischemia-reperfusion. Parallel studies in nondiabetic hearts showed that NKCC inhibition was not cardioprotective. These findings demonstrate that flux via NKCC is increased in type I diabetic hearts and that inhibition with bumetanide attenuates changes in Na(i) and ATP during ischemia and protects against ischemic injury. The data suggest a therapeutic role for pharmacological agents that inhibit flux via NKCC in diabetic patients with myocardial ischemia

Metabolic interventions that promote glucose use during ischemia have been shown to protect ischemic myocardium and improve functional recovery on reperfusion. We evaluated whether the cardioprotection afforded by high glucose during low-flow ischemia is associated with changes in the sarcolemmal content of glucose transporters, specifically GLUT-4. Isolated rat hearts were paced at 300 beats/min and perfused under normal glucose (5 mM) or high glucose (10 mM) conditions in buffer containing 0.4 mM albumin, 0.4 mM palmitate, and 70 mU/l insulin and subjected to 50 min of low-flow ischemia and 60 min of reperfusion. To determine the importance of insulin-sensitive glucose transporters in mediating cardioprotection, a separate group of hearts were perfused in the presence of cytochalasin B (10 microM), a preferential inhibitor of insulin-sensitive glucose transporters. Ischemic contracture during low-flow ischemia and creatine kinase release on reperfusion was decreased, and the percent recovery of left ventricular function with reperfusion was enhanced in hearts perfused with high glucose (P < 0.03). Hearts perfused with high glucose exhibited increased GLUT-4 protein expression in the sarcolemmal membrane compared with control hearts under baseline conditions, and these changes were additive with low-flow ischemia. In addition, high glucose did not affect the baseline distribution of sarcolemmal GLUT-1 and blunted any changes with low-flow ischemia. These salutary effects were abolished when glucose transporters are blocked with cytochalasin B. These data demonstrate that protection of ischemic myocardium by high glucose is associated with increased sarcolemmal content of the insulin-sensitive GLUT-4 and suggest a target for the protection of jeopardized myocardium