Faculty Bibliography

OBJECTIVE: We have previously demonstrated that zopolrestat, an inhibitor of the enzyme aldose reductase, reduces ischemic injury in hearts from diabetic and non-diabetic rats. To further explore potential cardioprotective mechanisms of zopolrestat, we measured changes in intracellular sodium, calcium, and Na+,K(+)-ATPase activity in zopolrestat treated hearts during ischemia and reperfusion. METHODS: Hearts from acute diabetic (Type I) and age-matched control rats were isolated and retrogradely perfused. Hearts had either control perfusion or exposure to 1 microM zopolrestat for 10 min, followed by 20 min of global ischemia and 60 min of reperfusion. Changes in intracellular sodium and calcium were measured using 23Na and 19F magnetic resonance spectroscopy, respectively, while the activity of Na+,K(+)-ATPase was measured using biochemical assays. RESULTS: Zopolrestat blunted the rise in [Na]i during ischemia in both diabetic hearts and non-diabetic hearts. The end-ischemic [Na]i was 21.3 +/- 2.6 mM in the zopolrestat treated diabetics and 25.9 +/- 2.3 in zopolrestat treated non-diabetics, versus 31.6 +/- 2.6 mM and 32.9 +/- 2.8 mM in the untreated diabetics and untreated non-diabetics, respectively, (P = 0.002). Similarly, the rise in [Ca]i at the end of ischemia was significantly reduced in zopolrestat treated diabetic and non-diabetic hearts (P = 0.005). Zopolrestat increased the activity of Na-,K(+)-ATPase in diabetic hearts under baseline conditions (11.70 +/- 0.95 versus 7.28 +/- 0.98 mumol/h/mg protein, P = 0.005) as well as during ischemia and reperfusion. Similar changes in Na+,K(+)-ATPase activity were also observed in non-diabetic hearts. CONCLUSIONS: The data provide additional support to the protective effects of zopolrestat and suggest that a possible mechanism of action may be associated with the attenuation of the rise in [Na]i and [Ca]i during ischemia and reperfusion

Lipoprotein lipase (LPL) is the central enzyme in plasma triglyceride hydrolysis. In vitro studies have shown that LPL also can enhance lipoprotein uptake into cells via pathways that are independent of catalytic activity but require LPL as a molecular bridge between lipoproteins and proteoglycans or receptors. To investigate whether this bridging function occurs in vivo, two transgenic mouse lines were established expressing a muscle creatine kinase promoter-driven human LPL (hLPL) minigene mutated in the catalytic triad (Asp156 to Asn). Mutated hLPL was expressed only in muscle and led to 3,100 and 3,500 ng/ml homodimeric hLPL protein in post-heparin plasma but no hLPL catalytic activity. Less than 5 ng/ml hLPL was found in preheparin plasma, indicating that proteoglycan binding of mutated LPL was not impaired. Expression of inactive LPL did not rescue LPL knock-out mice from neonatal death. On the wild-type (LPL2) background, inactive LPL decreased very low density lipoprotein (VLDL)-triglycerides. On the heterozygote LPL knock-out background (LPL1) background, plasma triglyceride levels were lowered 22 and 33% in the two transgenic lines. After injection of radiolabeled VLDL, increased muscle uptake was observed for triglyceride-derived fatty acids (LPL2, 1.7x; LPL1, 1.8x), core cholesteryl ether (LPL2, 2.3x; LPL1, 2.7x), and apolipoprotein (LPL1, 1.8x; significantly less than cholesteryl ether). Skeletal muscle from transgenic lines had a mitochondriopathy with glycogen accumulation similar to mice expressing active hLPL in muscle. In conclusion, it appears that inactive LPL can act in vivo to mediate VLDL removal from plasma and uptake into tissues in which it is expressed

Alterations in glucose metabolism have been implicated in the cardiovascular complications of diabetes. Previous work in this laboratory demonstrated that hearts from diabetic animals have an elevated cytosolic redox ratio (NADH/NAD+) and that this redox imbalance is probably due to elevated polyol pathway flux. We therefore hypothesized that 1) the elevated cytosolic redox ratio of diabetic hearts could result in inhibition of glycolytic enzymes sensitive to the redox state, 2) polyol pathway inhibition could restore the abnormal glucose metabolism of diabetic hearts, and 3) the relative incorporation of mixed substrates into hearts from diabetic animals would demonstrate less glycolytic and more fatty acid oxidation. Hearts from diabetic (BB/W) and nondiabetic control rats were perfused with buffers containing 13C-labeled substrates, and the metabolism of these hearts was analyzed using 13C NMR spectroscopy. Tissue samples were analyzed for metabolite levels using biochemical assay. Compared with controls, diabetic hearts had glyceraldeyde 3-phosphate levels that were four times greater than nondiabetic hearts and exhibited 91% less 13C labeling of lactate and 92% less 13C labeling of glutamate (P < 0.03). Aldose reductase inhibition with zopolrestat restored the metabolite labeling of diabetic hearts. Diabetic hearts perfused with a mixture of substrates used 53% more acetate than nondiabetic control hearts (P < 0.05), and aldose reductase inhibition lowered the acetate utilization of diabetic hearts by 9% (P < 0.05). These data suggest that glycolytic flux in diabetic hearts is inhibited at glyceraldehyde-3-phosphate dehydrogenase and that inhibition of the polyol pathway with zopolrestat increases glycolytic flux in these hearts. Furthermore, hearts from diabetic animals showed a marked dependence on fatty acids for substrate utilization compared with nondiabetic controls, consistent with inhibition of the pyruvate dehydrogenase complex in diabetic hearts

Several studies have shown that maintenance of glycolysis limits the metabolic and functional consequences of low-flow ischemia. Because diabetic animals are known to have impaired glycolytic metabolism coupled with increased flux through the aldose reductase (AR) pathway, we hypothesized that inhibition of AR would enhance glycolysis and thereby improve metabolic and functional recovery during low-flow ischemia. Hearts (n = 12) from nondiabetic control and diabetic rats were isolated and retrograde perfused using 11 mM glucose with or without the AR inhibitor zopolrestat (1 microM). Hearts were subjected to 30 min of low-flow ischemia (10% of baseline flow) and 30 min of reperfusion. 31P NMR spectroscopy was used to monitor time-dependent changes in phosphocreatine (PCr), ATP, and intracellular pH. Changes in the cytosolic redox ratio of NADH to NAD+ were obtained by measuring the ratio of tissue lactate to pyruvate. Effluent lactate concentrations and oxygen consumption were determined from the perfusate. AR inhibition improved functional recovery in both control and diabetic hearts, coupled with a lower cytosolic redox state and greater effluent lactate concentrations during ischemia. ATP levels during ischemia were significantly higher in AR-inhibited hearts, as was recovery of PCr. In diabetic hearts, AR inhibition also limited acidosis during ischemia and normalized pH recovery on reperfusion. These data demonstrate that AR inhibition maintains higher levels of high-energy phosphates and improves functional recovery upon reperfusion in hearts subjected to low-flow ischemia, consistent with an increase in glycolysis. Accordingly, this approach of inhibiting AR offers a novel method for protecting ischemic myocardium.

INTRODUCTION: Fasting increases myocardial glycogen content and has been shown to limit injury and improve recovery following no-flow ischaemia in the isolated heart. However, the protective role of glycogen loading per se in fed animals has been questioned by data in preconditioned animals showing that reduced glycogenolysis may be protective prior to no-flow ischemia. Therefore, we hypothesized that fasting protects the globally ischemic heart by mechanisms separate from glycogen loading. METHODS: Isolated hearts from rats fasted for 24 h were retrogradely perfused using glucose substrate and subjected to 20 min of global no-flow ischemia. Fed rats were identically perfused either under control conditions (glucose substrate) or with an intervention chosen to increase tissue glycogen (glucose plus insulin, [insulin]) prior to ischemia. Functional recovery and creatine kinase (CK) release were measured during reperfusion. Nuclear magnetic resonance spectroscopy was used to measure intracellular pH, phosphorylated glycolytic intermediates and high-energy phosphates, while the lactate and pyruvate contents of the hearts were measured prior to and at the end of ischemia. RESULTS: Heart from fasted animals had significantly increased glycogen content prior to ischemia (76.6 +/- 2 vs. 40.9 +/- 3 mumol glu/gdw in control hearts, P < 0.05) as did hearts exposed to insulin (88.6 +/- 10 mumol glu/gdw), but only hearts from fasted animals had greater glycogen utilization during ischemia. Hearts from fasted animals also had lower levels of lactate relative to pyruvate (L/P) under baseline conditions and, on reperfusion, reduced CK release (fasted: 183 +/- 48 versus control: 756 +/- 56 IU/gdw, P < 0.05). Conversely, insulin hearts had increased CK release (1831 +/- 190 IU/gdw, P < 0.001 vs control) and worse functional and metabolic recovery on reperfusion. Compared to the insulin hearts, hearts from fasted animals had both less acidosis and less rapid depletion of ATP during ischemia, as well as lower accumulation of glycolytic intermediates. CONCLUSION: Fasting protects the heart from ischemic injury and is associated with a lower L/P ratio and increased glycogen utilization during ischemia. In contrast, increasing glycogen content in hearts from fed animals using insulin limits glycogen utilization, increases ischemic injury, and impairs both functional and metabolic recovery under conditions of 20 min of global no-flow ischemia

INTRODUCTION: Pharmacologic inhibition of the Na-H exchanger prior to and during ischemia has been shown to protect the ischemic heart by reducing Na-H exchange. However, pH regulation in the ischemic heart is primarily mediated by other pH regulatory mechanisms, such as metabolite efflux and sodium-coupled HCO3-influx, which may compensate for a reduction in Na-H exchange by increasing proton efflux. We hypothesized that short-term pharmacologic inhibition of the Na-H exchanger would result in increases in other compensatory pH regulatory mechanisms and thereby limit acidosis during ischemia and reduce ischemic injury. METHODS: In order to test this hypothesis, we exposed isolated perfused rat hearts to ethylisopropylamiloride (EIPA, 3 micro M) for 40 min, followed by 10 min of EIPA-free perfusate and 30 min of global ischemia (termed CTL/EIPA hearts). The effects of this intervention were compared to hearts perfused with either glucose alone (CTL) or EIPA 3 micro M for 10 min before ischemia (EIPA). Ischemic injury was measured using creatine kinase (CK) release on reperfusion, while pH and metabolic effects were measured using 31P nuclear magnetic resonance spectroscopy. The effect of this intervention on recovery from an acid load was assessed using an NH4Cl pre-pulse in bicarbonate-containing Krebs-Henseleit as well as a HEPES buffer. RESULTS: Both CTL/EIPA and EIPA hearts had marked reduction in ischemic injury (CK control 1191 +/- IU/g dry weight: CTL/EIPA 406 +/- 42 IU/gdw; EIPA 333 +/- 78 IU/gdw), as well as significantly reduced end-diastolic pressure on reperfusion. Intracellular pH was higher in the CTL/EIPA hearts (end-ischemic pH = 6.34 +/- 0.05) compared to either control (5.86 +/- 0.02) or EIPA hearts (6.01 +/- 0.02), while pH recovery on reperfusion was markedly slowed in the CTL/EIPA hearts. CTL/EIPA hearts had rapid ATP depletion during ischemia, but PCr recovery comparable to EIPA hearts. Acidification on exposure to NH4Cl was increased in the presence of HEPES, but ph recovery was not altered by short-term exposure to EIPA. CONCLUSIONS: These data show that short-term inhibition of the Na-H altered pH regulation in the ischemic heart, resulting in reduced acidosis and slow pH recovery on reperfusion, coupled with reduction in ischemic injury and end-diastolic pressure on a reperfusion. These findings are consistent with short-term exposure to EIPA accelerating ATP depletion during ischemia, as well as limiting proton efflux during reperfusion

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

One or more brief periods of ischemia, termed preconditioning, dramatically limits infarct size and reduces intracellular acidosis during subsequent ischemia, potentially via enhanced sarcolemmal proton efflux mechanisms. To test the hypothesis that preconditioning increases the functional activity of sodium-dependent proton efflux pathways, isolated rat hearts were subjected to 30 min of global ischemia with or without preconditioning. Intracellular sodium (Nai) was assessed using 23Na magnetic resonance spectroscopy, and the activity of the Na-H exchanger and Na-K-2Cl cotransporter was measured by transiently exposing the hearts to an acid load (NH4Cl washout). Creatine kinase release was reduced by greater than 60% in the preconditioned hearts (P < 0.05) and was associated with improved functional recovery on reperfusion. Preconditioning increased Nai by 6.24 +/- 2.04 U, resulting in a significantly higher level of Nai before ischemia than in the control hearts. Nai increased significantly at the onset of ischemia (8.48 +/- 1.21 vs. 2.57 +/- 0.81 U, preconditioned vs. control hearts; P < 0.01). Preconditioning did not reduce Nai accumulation during ischemia, but the decline in Nai during the first 5 min of reperfusion was significantly greater in the preconditioned than in the control hearts (13.48 +/- 1.73 vs. 2.54 +/- 0.41 U; P < 0.001). Exposure of preconditioned hearts to ethylisopropylamiloride or bumetanide in the last reperfusion period limited in the increase in Nai during ischemia and reduced the beneficial effects of preconditioning. After the NH4Cl prepulse, preconditioned hearts acidified significantly more than control hearts and had significantly more rapid recovery of pH (preconditioned, delta pH = 0.35 +/- 0.04 U over 5 min; control, delta pH = 0.15 +/- 0.02 U over 5 min). This rapid pH recovery was not affected by inhibition of the Na-K-2Cl cotransporter but was abolished by inhibition of the Na-H exchanger. These results demonstrate that preconditioning alters the kinetics of Nai accumulation during global ischemia as well as proton transport after NH4Cl washout. These observations are consistent with stimulation of the Na-K-2Cl cotransporter and Na-H exchanger by preconditioning

Limitation of myocardial injury and infarction has been demonstrated by interventions such as ischemic preconditioning or the use of pyruvate as a substrate, which reduces glycogen content before, and acidosis during, ischemia. An isolated perfused rat heart model of global ischemia was employed to test the hypothesis that glycogen depletion reduces ischemic injury as measured by creatine kinase release. 31P-nuclear magnetic resonance spectroscopy was used to measure high-energy phosphates (ATP and phosphocreatine), phosphomonoesters (PME), and intracellular pH. Compared with control glucose-perfused hearts with normal glycogen content (1.49 +/- 0.13 mg Glc/g wet wt), glycogen-depleted pyruvate, ischemic preconditioned, and glycogen-depleted glucose hearts all had reduced glycogen content before ischemia (0.62 +/- 0.16, 0.81 +/- 0.10, and 0.67 +/- 0.12 mg Glc/g wet wt, respectively; P = 0.003) and significantly higher pH at the end of ischemia (5.85 +/- 0.02, 6.33 +/- 0.06, 6.24 +/- 0.04, and 6.12 +/- 0.02 in control, glycogen-depleted pyruvate, preconditioned, and glycogen-depleted glucose-perfused hearts, respectively; P < 0.01), although acidification during the initial phase of ischemia was differentially affected by the three interventions. Glycogen-depleted pyruvate and preconditioned hearts had reduced PME accumulation, greater recovery of function and phosphocreatine, and lower creatine kinase release on reperfusion, whereas glycogen-depleted glucose-perfused hearts were similar to control hearts. In summary, glycogen depletion by these three methods limits the fall in pH during global ischemia, although glycogen depletion in the absence of preconditioning does not limit ischemic injury.(ABSTRACT TRUNCATED AT 250 WORDS)