INTRODUCTION

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POSTISCHEMIC MYOCARDIAL STUNNING is produced in large part by cytotoxic oxygen and nitrogen metabolites generated upon coronary reperfusion (6). The resulting superoxide, H₂O₂, hydroxyl radical, and peroxynitrite (2, 12, 39) modify a variety of cellular components (5) to produce stunning. Inotropic responses to -adrenergic stimulation are dampened in stunned myocardium (38, 44), perhaps resulting from oxidative damage to protein components of the -adrenergic signaling cascade.

Pyruvate, a natural metabolic fuel in myocardium, markedly increased -adrenergic responsiveness of stunned myocardium and preserved energy stores (44). Pyruvate also restored GSH/GSSG redox potential (43), the principal intracellular antioxidant system, and bolstered NADPH/NADP⁺ redox potential, the source of reducing power to regenerate GSH from GSSG (29). N-acetylcysteine, a membrane-permeable antioxidant, recapitulated the pyruvate enhancement of -adrenergic inotropism despite the failure of this nonfuel to prevent -adrenergic depletion of myocardial energy reserves. Like pyruvate, N-acetylcysteine increased GSH redox potential. These combined findings indicated that pyruvate's antioxidant actions, more than its enhancement of energy reserves, mediated its restoration of -adrenergic inotropism in stunned myocardium (43).

We recently reported that acetoacetate sharply increased GSH redox potential and contractile performance of myocardium challenged by H₂O₂ in the absence of ischemia (42). If acetoacetate exerts similar antioxidant actions in stunned myocardium, then it could potentiate -adrenergic inotropism as did pyruvate and N-acetylcysteine. To test this proposal, postischemic, stunned guinea pig hearts were treated with 5 mM acetoacetate alone or in combination with isoproterenol at a low concentration (2 nM) that only modestly increases contractile performance in the absence of antioxidants (43, 44). The impact of these treatments on left ventricular contractile performance was compared with their effects on myocardial energy reserves, antioxidant redox potential, and cAMP. This study demonstrated that acetoacetate only modestly increased contractile performance of stunned myocardium but powerfully potentiated inotropic responses to isoproterenol. These contractile responses paralleled acetoacetate's augmentation of GSH redox potential, which was potentiated by isoproterenol coadministration.

	MATERIALS AND METHODS

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Isolated working hearts. Animal experimentation was approved by the Animal Care and Use Committee of the University of North Texas Health Science Center and was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH, 1996). Hearts (n = 69) were excised from male Hartley guinea pigs (400-600 g) and antegradely perfused as working organs with Krebs-Henseleit bicarbonate buffer (44). All perfusion media were maintained at 38°C, aerated with 95% O₂-5% CO₂, and fortified with 10 mM glucose. Heart rate, aortic pressure (P_a), left atrial filling pressure (P_v), and cardiac output (sum of aortic and coronary flows) were measured to determine cardiac function (8). Left ventricular function was assessed from developed pressure (i.e., P_a  P_v) × heart rate (HR) (cmH₂O/min), stroke work (mJ/g), and power (mJ · min¹ · g¹), which equaled stroke work times HR.

Ischemia-reperfusion protocol to produce cardiac stunning. After 15 min of preischemic baseline perfusion, hearts were subjected to 45 min coronary underperfusion by lowering P_a and were concomitantly stimulated with 0.4 µM l-norepinephrine (44). Ischemic hearts were reperfused by restoring P_a to 90 cmH₂O and discontinuing l-norepinephrine infusion. P_a subsequently declined before stabilizing by 10-15 min of reperfusion at 40-50 cmH₂O, reflecting contractile impairment typical of myocardial stunning (44). P_v was held at 10-12 cmH₂O throughout the protocol.

Metabolic and -adrenergic treatment of stunned hearts. The following four groups of stunned hearts were examined: no treatment (n = 12), 5 mM acetoacetate at 15-45 min of reperfusion (n = 10), 2 nM isoproterenol at 30-45 min of reperfusion (n = 8), or combined treatment with 5 mM acetoacetate at 15-45 min and 2 nM isoproterenol at 30-45 min (n = 9). Contractile performance and metabolic state of these stunned hearts were compared with nonischemic time control hearts perfused for 105 min, either without treatment (n = 12) or treated with 2 nM isoproterenol at 90-105 min (n = 6). Isoproterenol stock solution (100 nM) was freshly prepared 10-15 min before infusion in 0.9% NaCl-1% ascorbic acid and shielded from light to prevent autooxidation. Sucrose was continuously infused to achieve a 2 mM left atrial concentration during the final 5 min to determine extracellular space, as recently described (44). All hearts were freeze clamped with liquid N₂-precooled Wollenberger tongs and stored at 90°C before metabolite extraction.

Acetoacetate infusion/washout studies. Additional experiments were conducted to determine whether acetoacetate's impact on contractile performance, -adrenergic inotropism, and GSH redox potential of stunned myocardium persisted after acetoacetate was discontinued. Acetoacetate (5 mM) was infused during 15-45 min reperfusion, and 2 nM isoproterenol (n = 6) or its NaCl/ascorbic acid vehicle (n = 6) was infused during 30-60 min reperfusion. Hearts were freeze-clamped at 60 min reperfusion, after 15 min acetoacetate washout.

Myocardial metabolites. Frozen hearts were pulverized in a precooled porcelain mortar under liquid nitrogen. The powdered tissue was extracted (18, 27) for measurement of ATP, phosphocreatine (PCr), creatine (Cr), P_i, citrate, glucose-6-phosphate, NADP⁺, NADPH, GSH, GSSG, and sucrose. Metabolites were assayed (4) in a Shimadzu model UV-1601PC dual-wavelength uv/vis spectrophotometer (337 nm measuring wavelength, 417 nm reference wavelength,  = 5.65 M¹ · cm¹). Intracellular P_i was determined by subtracting extracellular P_i, i.e., perfusate P_i concentration times sucrose distribution volume, from total myocardial P_i content (27, 33). PCr phosphorylation potential {[PCr]/([Cr][P_i])} was calculated as an index of cellular energy state (44). GSH and GSSG were measured according to Akerboom and Sies (1). Myocardial cAMP content was determined by RIA (28), as recently described (44).

Statistical analyses. Data are expressed as means ± SE. Cardiac performance was analyzed using two-way ANOVA. Metabolites, phosphorylation potential, and GSH and NADPH redox states in the different groups were compared by one-way ANOVA. Within-group comparisons at different times in the experiment were accomplished with one-way ANOVA for repeated measures. When ANOVA detected significant differences, the Student-Newman-Keul's multiple-comparison test was used post hoc to identify the specific differences. Statistical significance was assumed at P < 0.05.

	RESULTS

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Cardiac function. Contractile responses to 2 nM isoproterenol and/or 5 mM acetoacetate were determined in postischemic hearts. In stunned nontreated hearts, cardiac power stabilized at ~10% of the nonischemic time control value (Fig. 1), indicating severe contractile impairment of postischemic myocardium. Acetoacetate or isoproterenol alone increased power five- and fourfold, respectively, vs. pretreatment baseline. Combined acetoacetate plus isoproterenol treatment elicited a much more robust response that far exceeded the sum of the individual treatment effects; here, cardiac power increased 16-fold, to 156% of the time control value. Thus the combined metabolic and -adrenergic treatments elicited a powerful, synergistic enhancement of cardiac performance.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Cardiac power: impact of stunning and treatment. Values in Figs. 1-8 are means ± SE. Power was determined at 45 min reperfusion. Hearts received acetoacetate (AcAc) between 15 and 45 min and isoproterenol (ISO) between 30 and 45 min reperfusion. Values in untreated stunned (NoTx) and nonischemic time control (TC) hearts were obtained at the same perfusion times as the treatment values. *P < 0.05 vs. TC; P < 0.05 vs. all other groups.

Kinetics of isoproterenol response. Figure 2 compares the time course of the contractile response of nonischemic time control and stunned and acetoacetate-treated stunned hearts to 2 nM isoproterenol. The robust inotropic response of time control hearts reached a plateau within 5 min. Without acetoacetate treatment, the stunned hearts were much less responsive to the same concentration of isoproterenol. In these stunned hearts, the inotropic response to isoproterenol reached a reduced plateau within 10 min. Thus the response to adrenergic stimulation was blunted, not merely delayed. Although acetoacetate pretreatment produced only a moderate increase in cardiac power, the same treatment produced a substantial increase in the inotropic response to isoproterenol.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Time course of Iso's inotropic actions: impact of stunning and AcAc. Stunned hearts were treated with 2 nM Iso in the absence () and presence () of 5 mM AcAc. Time-control hearts () were perfused for 90 min without ischemia or AcAc before Iso infusion. Bars at top indicate the periods of AcAc and Iso administration. *P < 0.05 vs. 15 min of reperfusion. P < 0.05 vs. 15 and 30 min of reperfusion.

cAMP. The intracellular second messenger for -adrenergic signaling, cAMP, was measured to determine the impact of stunning and postischemic treatments on the signaling mechanism. Neither stunning per se nor treatment of stunned myocardium with 2 nM isoproterenol altered cAMP content vs. time control myocardium (Fig. 3). Treatment with acetoacetate alone unexpectedly depleted cAMP by 30% vs. untreated or isoproterenol-treated stunned myocardium. In contrast, treatment with acetoacetate in combination with isoproterenol increased cAMP content significantly above all other groups. Indeed, the combined treatments increased cAMP content by 30% over treatment with isoproterenol alone, indicating acetoacetate-potentiated -adrenergic signaling.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Myocardial cAMP content. cAMP was measured in hearts snap frozen at 45 min postischemia or at 105 min of time-control perfusion. *P < 0.05 vs. NoTx; P < 0.05 vs. ISO; P < 0.05 vs. all groups.

Myocardial phosphorylation potential. Myocardial energy state was assessed from PCr phosphorylation potential {[PCr]/([Cr][P_i])}, a measure of cytosolic ATP phosphorylation potential (46). PCr potential of the untreated stunned myocardium was similar to the time control value (Fig. 4), despite the disparity in contractile performance of these two groups. Acetoacetate tended to increase, and isoproterenol to lower, phosphorylation potential vs. stunned myocardium, although neither effect was significant. Moreover, phosphorylation potential of the acetoacetate hearts was 70% greater than that of isoproterenol-treated hearts (P < 0.01) at similar levels of mechanical performance (Fig. 1). The combination of acetoacetate with isoproterenol prevented further decline in myocardial energy state (Fig. 4) despite the severalfold increase in contractile function (Figs. 1 and 2).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Myocardial phosphorylation potential. Myocardial phosphocreatine (PCr) and creatine (Cr) content and inorganic phosphate concentration ([Pi]i) were determined in the same experiments as in Figs. 1 and 2. *P < 0.05 vs. ISO.

GSH redox potential. Components of the GSH/NADPH redox system were measured to determine the impact of metabolic and inotropic treatments on antioxidant redox potential of stunned myocardium. GSH content tended to be lower in untreated stunned vs. time control myocardium, but the difference was not statistically significant. GSSG doubled, and the GSH-to-GSSG ratio, a measure of cellular antioxidant redox potential (40), fell 71% in stunned vs. time control myocardium (Fig. 5). Treatment with acetoacetate alone lowered GSSG content 68% and quadrupled GSH/GSSG redox potential in stunned myocardium. Isoproterenol also lowered GSSG, although not as much as acetoacetate, and tripled GSH/GSSG. Remarkably, when combined, the two interventions powerfully increased the GSH/GSSG redox state to double the time control value. Myocardial GSH content was similar among the treatments, so GSH/GSSG redox potential was altered in each case by changes in GSSG content alone.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   GSH content and redox state. Myocardial GSH and GSSG contents were measured in the same experiments as in Figs. 1-4. *P < 0.05 vs. all other groups.

View larger version (37K): [in this window] [in a new window]   Fig. 6.   Myocardial NADPH and NADP+. NADPH and NADP+ were measured in the same hearts as in Figs. 1-5. *P < 0.05 vs. TC; P < 0.05 vs. NoTx; P < 0.05 vs. ISO.

Citrate and glucose-6-phosphate. NADPH is generated in myocardium by two metabolic mechanisms mediated by citrate. Citrate accumulation could promote flux through the cytosolic, NADP⁺-specific isocitrate dehydrogenase reaction by providing substrate for aconitase-catalyzed isocitrate formation. Second, citrate could increase NADPH formation by inhibiting phosphofructokinase (19), which would divert glycolytic flux into the NADPH-generating hexose monophosphate shunt. In the latter scenario, glucose-6-phosphate, a glycolytic intermediate proximal to phosphofructokinase, would accumulate and provide substrate for the hexose monophosphate pathway. To test the possibility that acetoacetate and/or isoproterenol activated NADPH-generating pathways, citrate and glucose-6-phosphate were measured in stunned and time control hearts. Contents of these compounds did not differ in stunned vs. time control myocardium (Fig. 7). Given separately, acetoacetate and isoproterenol tended to double citrate content and increase glucose-6-phosphate content by roughly 35-45%, but only the acetoacetate-induced increase in glucose-6-phosphate was statistically significant vs. untreated stunned myocardium. In combination, the two treatments increased citrate fourfold and doubled glucose-6-phosphate content vs. time control and untreated stunned myocardium. Thus acetoacetate, in combination with isoproterenol, powerfully increased myocardial citrate and glucose-6-phosphate contents, bolstering substrate supply for pathways generating NADPH reducing power.

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Citrate and glucose-6-phosphate contents. Metabolites were measured in the same experiments as in Figs. 1-6. *P < 0.05 vs. all other groups.

Persistence of -adrenergic potentiation. The persistence of acetoacetate's improvements of cardiac function, -adrenergic inotropism, and GSH redox state was tested by discontinuing acetoacetate treatment while maintaining infusion of isoproterenol or vehicle. In the absence of isoproterenol, the acetoacetate enhancement of cardiac power reached a plateau by 30 min treatment but gradually subsided after acetoacetate was discontinued (Fig. 8A). The time course of the postacetoacetate decline in power resembled that of the antecedent power increase. These relationships are clearly illustrated by plotting power as a percentage of the peak value achieved during treatment (Fig. 8B). In contrast, the robust contractile response to isoproterenol in the acetoacetate-treated stunned hearts declined slightly during acetoacetate washout (Fig. 8A). In fact, when the increase in power produced by acetoacetate alone was subtracted from the total power during the combined treatment, the difference, i.e., the "isoproterenol effect" (Fig. 8A), remained at its peak value after acetoacetate was discontinued. Thus restored -adrenergic inotropism persisted beyond the period of acetoacetate treatment. In contrast, GSH redox state, which was increased by acetoacetate and especially by the combination of acetoacetate and isoproterenol (Fig. 5), returned to baseline by 15 min postacetoacetate, even when isoproterenol infusion was maintained (Fig. 8C). Thus the temporary enhancement of GSH redox potential by acetoacetate appears to have produced a more persistent restoration of -adrenergic inotropism in stunned myocardium.

View larger version (28K): [in this window] [in a new window]   Fig. 8.   Effect of AcAc washout on contractile function and glutathione redox potential of stunned myocardium in absence vs. presence of Iso. A and B: data from untreated (; n = 12), 5 mM AcAc + NaCl/ascorbic acid vehicle-treated (; n = 6), and AcAc + 2 nM Iso-treated (; n = 6) stunned hearts. AcAc and Iso or its vehicle was administered between 15 and 45 and between 30 and 60 min of reperfusion, respectively. The effect of Iso on cardiac power in the AcAc + Iso-treated hearts (ISO effect: ) was determined by subtracting the mean AcAc-induced increase in power in the AcAc + vehicle group (i.e., AcAc + vehicle power  untreated power) from power in each AcAc + Iso experiment. In B, power in the AcAc + vehicle and AcAc + ISO groups is normalized as a percentage of the peak power achieved in each experiment. C: glutathione redox state ([GSH]/[GSSG]) at 45 and 60-min reperfusion, i.e., at 30-min AcAc treatment and 15-min AcAc washout. A: *P < 0.05 vs. 15 min reperfusion; P < 0.05 vs. 30 min reperfusion; P < 0.05 vs. 45 min reperfusion. C: *P < 0.05 vs. 45-min value in the same treatment group; P < 0.05 vs. AcAc + vehicle value at the same time.

	DISCUSSION

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Recently, pyruvate's antioxidant properties were shown to potentiate -adrenergic inotropism and cAMP formation in postischemic stunned myocardium (44). Pyruvate appeared to enhance -adrenergic inotropism by augmenting GSH and NADPH antioxidant redox potentials (43). This antioxidant effect may have restored the redox status of proteins in the -adrenergic signaling cascade. The ketone body acetoacetate, a natural energy-yielding fuel in myocardium, increased GSH/GSSG and contractile performance of H₂O₂ injured myocardium (42). Thus acetoacetate, like pyruvate, might increase contractile performance and potentiate -adrenergic inotropism in stunned myocardium by antioxidant mechanisms.

Stunned myocardium was much less responsive to 2 nM isoproterenol than nonischemic control myocardium, as previously reported (44). The increase in cardiac power produced by this moderate isoproterenol dose fell by 74% after stunning. Acetoacetate increased contractile function of the stunned myocardium. Although modest, this enhanced function was similar to that observed with the same concentration of pyruvate in the postischemic myocardium (44). Responses of stunned myocardium to isoproterenol were dramatically enhanced after pretreatment with acetoacetate. Indeed the combination of acetoacetate and isoproterenol produced a four- to sixfold increase in cardiac power compared with either acetoacetate or isoproterenol alone. Thus acetoacetate, like pyruvate (44), dramatically improved -adrenergic inotropism in the stunned myocardium.

GSH redox potential and cAMP formation. The GSH system, the central element of the myocardium's endogenous antioxidant defenses (41), neutralizes peroxides (14) and peroxynitrite (12) to prevent oxidative damage. GSH also restores oxidized protein sulfhydryls to their reduced state (16) and thereby maintains the catalytic activity of enzymes susceptible to oxidative stress (11, 35). Myocardial stunning reduces the heart's sensitivity to -adrenergic stimulation by lowering the binding affinity and density of ₁-adrenergic receptors and by inactivating adenylate cyclase (38) through the oxidation of sulfhydryl groups within the enzyme (15). Indeed, H₂O₂, which has been implicated in the pathogenesis of cardiac stunning (17, 26), impaired -adrenergic signaling in nonischemic hearts by lowering the density and affinity of ₁-adrenoceptors, altering G_s protein function, and blunting basal- and isoproterenol-stimulated adenylate cyclase activity (36, 37). GSH redox potential fell by 71% in stunned myocardium, indicating appreciable oxidative stress resulting from ischemia/reperfusion. Isoproterenol increased GSH redox potential modestly, and acetoacetate restored GSH potential to the time control level. Once again, the combined interventions increased GSH redox potential well above either agent alone. By augmenting the reducing power of the GSH system, acetoacetate may have helped restore sulfhydryls of proteins inactivated by oxidative stress, including -adrenergic signaling proteins.

Citrate, glucose-6-phosphate, and antioxidant redox potential. Acetoacetate may enhance GSH/GSSG by increasing myocardial citrate, which promotes metabolic flux through two NADPH-generating pathways. First, the conversion of citrate to isocitrate supplies substrate to the NADP⁺-dependent isocitrate dehydrogenase reaction (3). Second, citrate inhibits phosphofructokinase (19), causing glucose-6-phosphate to accumulate and diverting glycolytic flux into the NADPH-generating hexose monophosphate shunt. Acetoacetate alone moderately increased myocardial citrate and glucose-6-phosphate contents and the NADPH/NADP⁺ redox state. The combination of isoproterenol and acetoacetate increased these variables more substantially, possibly because of isoproterenol-activated glycogenolysis and glucose uptake (21, 22), which, when combined with citrate inhibition of phosphofructokinase (19), could powerfully increase NADPH formation in the hexose monophosphate shunt.

Impact of phosphorylation potential vs. GSH redox state on cardiac function and -adrenergic inotropism. Acetoacetate's enhancement of postischemic function and its potentiation of -adrenergic inotropism are strikingly similar to the actions of pyruvate in this model of myocardial stunning (44). Pyruvate increases PCr phosphorylation potential in parallel with contractile performance, leading to the proposal that this energetic enhancement could augment cardiac function by increasing substrate supply to the ATPases that orchestrate the cardiac cycle (7, 8, 10, 31, 32, 48). However, the present findings suggest modification of this working hypothesis, because pyruvate's antioxidant actions could be central to its cardiotonic effect. Acetoacetate did not increase phosphorylation potential of stunned myocardium, yet it increased preisoproterenol power just as much as pyruvate (44). Acetoacetate and pyruvate both potentiated the inotropic effect of isoproterenol to a similar degree [150 ± 10 vs. 108 ± 22 mJ · g¹ · min¹ (44)]. Both pyruvate and acetoacetate also increased GSH redox potential. Moreover, N-acetylcysteine, a pharmacological antioxidant that doesn't provide fuel for oxidative metabolism, also potentiated isoproterenol's inotropic effects despite a decline in phosphorylation potential (43). The enhancement of the GSH antioxidant system appears to be the principal mechanism of -adrenergic potentiation by metabolic fuels.

Acetoacetate appears to maintain GSH redox potential by additional, citrate-independent mechanisms. Acetoacetate and pyruvate were equally effective at restoring GSH/GSSG, although citrate content (µmol/g dry wt) in myocardium treated with pyruvate (8.6 ± 0.8) or pyruvate plus isoproterenol (4.8 ± 0.4; see Ref. 43) exceeded citrate contents in the respective acetoacetate groups (1.0 ± 0.2 and 1.9 ± 0.3; Fig. 7). Peroxynitrite and H₂O₂ are generated in postischemic myocardium and rank among the most important prooxidant mediators of cardiac stunning (6, 12). Both compounds convert GSH to GSSG and lower GSH/GSSG redox state: peroxinitrite directly oxidizes GSH (12, 25), and GSH is consumed by glutathione peroxidase to detoxify H₂O₂ (14, 29). Alternatively, acetoacetate can consume peroxynitrite in a direct aliphatic nitration reaction, yielding a nonreactive derivative, 2-nitroacetoacetate (45). Acetoacetate conversion to acetoacetyl-CoA by 3-oxoacid-CoA transferase (34) may also lessen oxidative stress. Acetoacetyl-CoA detoxifies H₂O₂, generating an unknown reactive oxygen intermediate that is subsequently neutralized by electrons donated by NADH (24). Both of these mechanisms would lower the prooxidant burden on the GSH system, facilitating recovery of the GSH/GSSG redox potential.

Persistent enhancement of -adrenergic inotropism after acetoacetate treatment. Once -adrenergic inotropism was restored by acetoacetate, it remained robust even after acetoacetate treatment was discontinued, despite the declining GSH redox state. In fact, the small loss in power attributed to discontinuing acetoacetate in the absence of isoproterenol quantitatively accounted for the same decline in power of the isoproterenol-treated hearts. Acetoacetate appears to have augmented endogenous antioxidant defenses and thus reversed oxidant injury to components of the -adrenergic signaling mechanism. Once these components were restored, continued augmentation of the GSH system was no longer required to maintain the signaling proteins.

Limitations. Although the decline in the GSH-to-GSSG ratio indicated prooxidant stress in postischemic myocardium, neither the specific prooxidant species nor the biomolecular targets of these compounds were identified or quantified in this investigation. Metabolites were measured in snap-frozen myocardium and expressed as total tissue content, without assessing intracellular metabolite compartmentation. This limitation is most problematic for metabolites like GSH, which are sequestered in separate, largely independent mitochondrial and cytosolic pools (23).