- Email: [email protected]
Insulin improves functional and metabolic recovery of reperfused working rat heart
Insulin Improves Functional and Metabolic Recovery of Reperfused Working Rat Heart Torsten Doenst, MD, R. Todd Richwine, MS, Molly S. Bray, PhD, Gary W. Goodwin, PhD, O. H. Frazier, MD, and Heinrich Taegtmeyer, MD, DPhil Division of Cardiology, Department of Medicine, and Division of Cardiothoracic Surgery, Department of Surgery, The University of Texas-Houston Medical School, Houston, Texas
Background. Glucose, insulin, and potassium solution improves left ventricular function in refractory pump failure. Direct effects of insulin on the heart cannot be determined in vivo. We hypothesized that insulin has a direct positive inotropic effect on the reperfused heart. Methods. Isolated working rat hearts were perfused with buffer containing glucose (5 mmol/L) plus oleate (1.2 mmol/L). Hearts were subjected to 15 minutes of ischemia and reperfused with or without insulin (100 mU/mL) for 40 minutes. Epinephrine (1 mmol/L) was added for the last 20 minutes. Results. Hearts recovered 51.1% of preischemic cardiac power in the absence and 76.4% in the presence of insulin (p < 0.05). Whereas oleate oxidation remained
unchanged, glucose uptake and oxidation increased during reperfusion with epinephrine (p < 0.01). This increase was significantly greater when hearts were reperfused in the presence of insulin (p < 0.01). Insulin also prevented an epinephrine-induced glycogen breakdown during reperfusion (p < 0.05). Conclusions. Insulin has a direct positive inotropic effect on postischemic rat heart. This effect is additive to epinephrine and occurs without delay. Increased rates of glucose oxidation and net glycogen synthesis are more protracted.
T
tion of the ischemic and reperfused myocardium are not known. It is not even certain whether insulin and enhanced glucose substrate metabolism affect the contractile function of the heart. The cardiac effects of GIK could be mediated by the systemic effects of insulin, which lower free fatty acid levels in the plasma and decrease systemic vascular resistance. With the advent of new methods of myocardial preservation and reperfusion of previously ischemic myocardium, the concept of substrate selection and metabolic support for the reperfused myocardium deserves to be reexamined in light of newer concepts of energy transfer in heart muscle. Ischemia is characterized by the depletion of glycogen and citric acid cycle intermediates [6]. Reperfusion metabolism is characterized by high rates of long-chain fatty acid oxidation, impaired glucose oxidation, and the uncoupling of energy production and utilization [7]. Even when glucose is the only substrate added to the perfusate, glucose utilization is depressed during reperfusion in the isolated working rat heart [8]. Regardless of the specific mechanism, there is reasonable support for the hypothesis that myocardial substrate metabolism per se has a direct effect on the contractile function of the postischemic, reperfused heart, which is mediated by enhanced rates of glucose oxidation. Glucose uptake and oxidation are, however, inhibited by long-chain fatty acids. Insulin may overcome the inhibition of glucose metabolism by mechanisms not yet completely understood. We tested the hypothesis that insulin has a direct positive inotropic effect on the reperfused heart and that this effect is mediated by increased rates of
he present study was undertaken to test the efficacy of insulin for recovery of function and glucose metabolism in the postischemic heart. The starting point was our observation that infusion of glucose, insulin, and potassium (GIK) solution improves contractile performance and reduces morbidity in patients with refractory heart failure after cardiopulmonary bypass [1]. Although the concept of metabolic support for the ischemic heart reperfused with GIK is not new, the rationale for the “polarizing solution” [2] was that insulin stimulates K1 reuptake and reduces the development of arrhythmias. However, some investigators have pointed out that enhanced rates of glycolysis result in H1 accumulation and impair contractile function during reperfusion [3], whereas others have observed that glycolysis preserves energy flux and contractile function of the heart during ischemia and reperfusion [4]. Clinical trials with GIK have shown a significant reduction in mortality of patients treated with GIK either in the course of an acute myocardial infarction [5] or in the course of refractory left ventricular failure after hypothermic ischemic arrest for aortocoronary bypass graft surgery [1]. Although the clinical implications of improved energy substrate metabolism seem obvious, the mechanisms by which alterations of metabolism can benefit the contracAccepted for publication Dec 16, 1998. Address reprint requests to Dr Taegtmeyer, Division of Cardiology, Department of Internal Medicine, University of Texas-Houston Medical School, 6431 Fannin, 1.246 MSB, Houston, TX 77030; e-mail: [email protected].
© 1999 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
(Ann Thorac Surg 1999;67:1682– 8) © 1999 by The Society of Thoracic Surgeons
0003-4975/99/$20.00 PII S0003-4975(99)00326-4
Ann Thorac Surg 1999;67:1682– 8
DOENST ET AL INOTROPIC EFFECTS OF INSULIN DURING REPERFUSION
1683
glucose oxidation, increased myocardial glycogen levels, and replenishment (anaplerosis) of the citric acid cycle. We used the isolated working rat heart perfused with physiologic concentrations of glucose and oleate, subjected it to total, global ischemia, and exposed it to epinephrine during reperfusion to mimic the clinical situation of reperfusion after controlled ischemic arrest of the heart.
Material and Methods Animals Male Sprague-Dawley rats (300 to 400 g) were obtained from Harlan (Indianapolis, IN) and housed in the University of Texas-Houston Medical School Animal Care Center. All animals had free access to food and water until the time of the experiment. All experiments were performed under protocols approved by The University of Texas-Houston Medical School. Animals received humane care in compliance with the “Guide for the Care and Use of Laboratory Animals” (NIH publication 85-23, revised 1985).
Materials d-[U-14C]glucose was obtained from ICN Biochemicals (Costa Mesa, CA). d-[2-3H]glucose was from Amersham Corp (Arlington Height, IL). [9,10-3H]oleate was from Sigma Chemical Co (St. Louis, MO). The purity of the 3 H-labeled tracers were ascertained by measuring the intrinsic 3H2O content. Fatty acid-free bovine serum albumin was obtained from Intergen Co (Purchase, NY). Enzymes for metabolite assays were from Boehringer Mannheim (Indianapolis, IN). All other chemicals were purchased from Sigma.
Working Heart Preparation The preparation has been described in detail earlier [9]. The perfusion apparatus has been modified to allow the collection of 14CO2 [10]. The apparatus was rendered gas tight and the exhaust was bubbled through a solution of 0.33 mmol/L benzethonium hydroxide for the collection of 14CO2 in the gaseous phase. Hearts were perfused as working hearts at 37°C with recirculating KrebsHenseleit buffer (200 mL) containing glucose (5 mmol/L) plus sodium-oleate (1.2 mmol/L) bound to 3% bovine serum albumin, Cohn fraction V, fatty acid-free, and equilibrated with 95% O2–5% CO2. Total perfusate [Ca21] was 2.5 mmol/L. All experiments were carried out with a preload of 15 cm H2O and an afterload of 100 cm H2O. The hearts were beating spontaneously. Aortic flow and coronary flow were measured every 5 minutes. Heart rate as well as systolic and diastolic aortic pressures were measured continuously with a 3F Millar transducer (Millar Instruments, Houston, TX) and a MacLab physiologic recording system (ADInstruments, Milford, MA).
Perfusion Protocol The perfusion protocol is shown in Figure 1. Hearts were perfused for 20 minutes under baseline conditions. Total,
Fig 1. Schematic of the experimental protocol and substrates used. Isolated working rat hearts were perfused with Krebs-Henseleit buffer at a preload of 15 cm H2O and an afterload of 100 cm H2O. See Material and Methods for details.
global, normothermic ischemia was induced for 15 minutes by clamping, and reperfusion was induced by unclamping, both the aortic and the atrial line of the perfusion system. In the experimental group, insulin (100 mU/mL) was added at the beginning of reperfusion (n 5 11, n 5 7 with [2-3H]glucose as tracer, n 5 4 with [U-14C]glucose and [9,10-3H]oleate as tracers). The control group (n 5 12, n 5 8 with [2-3H]glucose as tracer, n 5 4 with [U-14C]glucose and [9,10-3H]oleate as tracers) was reperfused without insulin. In both groups epinephrine (1 mmmol/L) was added after 20 minutes of reperfusion. Hearts were freeze-clamped 20 minutes after the addition of epinephrine. The use of the different tracer protocols did not affect contractile function or glycogen content of the hearts. In separate experiments, hearts were perfused according to the same protocol but were freeze-clamped before ischemia (n 5 4), at the end of ischemia (n 5 4), or after 20 minutes of reperfusion (n 5 5 with insulin, n 5 5 without insulin) for the determination of metabolites. Cardiac performance of these hearts was not different from those perfused for the entire duration of the experimental protocol (data not shown).
Assessment of Contractile Performance Cardiac performance was expressed as cardiac power (the product of cardiac output and mean aortic pressure) in terms of milliwatts as described earlier [11]. Mean aortic pressure (cm H2O) was calculated as (systolic 1 diastolic pressure 3 2)/3. Heart rate was measured as beats per minute and cardiac output as milliliters per minute. We used the average value of cardiac power during the last 10 minutes of the preischemic period as the reference value (baseline) for postischemic cardiac performance. Recovery of power during reperfusion was assessed separately for the time period before and after the addition of epinephrine. Recovery was determined as the mean value of all cardiac power measurements
1684
DOENST ET AL INOTROPIC EFFECTS OF INSULIN DURING REPERFUSION
during the first 20 minutes of reperfusion (early reperfusion) and during the last 20 minutes of reperfusion.
Ann Thorac Surg 1999;67:1682– 8
Table 1. Cardiac Power Before Ischemia, During Early Reperfusion, and Late Reperfusion With Epinephrinea Cardiac Power (mW)
Assessment of Uptake and Oxidation Rates Dual-label counting of 3H and 14C was performed on a Packard 1900 TR liquid scintillation analyzer by the method of spectral index analysis as described by the manufacturer (Packard Instruments, Meriden, CT). Glucose uptake and oleate oxidation were determined by the rate of 3H2O production from [2-3H]glucose and [9,10-3H]oleate, respectively [10, 11]. Release of 3H2O into the perfusate was analyzed in 5-minute intervals. 3H2O was separated from [2-3H]glucose or [9,10-3H]oleate in the perfusate by anion exchange chromatography on AG-1X8 resin (BioRad Laboratories, Hercules, CA) [10]. The amount of 3H2O in the perfusate was plotted against time, and the slopes of the desired intervals were used to calculate glucose uptake or oleate oxidation rates, which were expressed as micromoles per minute per gram of dry weight. Glucose oxidation was determined from the cumulative production of 14CO2 from [U-14C]glucose. The amount of 14CO2 present in both the exhaust and the perfusate was assessed. The exhausted 14CO2 was collected during 5-minute intervals by bubbling through 5 mL of 0.33 mmol/L benzethonium hydroxide in methanol. The content of 14CO2 in the perfusate was determined by trapping 14CO2 liberated on acidifying 500 mL of the perfusate samples in 1 mL of 1 mmol/L benzethonium hydroxide in methanol. Quantitative recovery of 14 CO2 was verified in a preliminary experiment by injecting a bolus of [14C]NaHCO3 (2 mCi) into the sealed perfusion apparatus and calculating recovery after determining 14CO2 in exhaust and perfusate. The sum of 14 CO2 in the perfusate and the exhaust was plotted against time, and the slopes of the desired intervals were used to calculate glucose oxidation rates, which were expressed as micromoles per minute per gram of dry weight.
Observation Period Before ischemia (0 –20 min) Early reperfusion (35–55 min) Late reperfusion (55–75 min)
No Insulin
Insulin During Reperfusion
8.54 6 0.90 (100%) 4.13 6 0.42 (51.1% 6 5.18%) 7.49 6 0.64 (92.6% 6 10.6%)
8.76 6 0.54 (100%) 6.89 6 0.78b (76.4% 6 4.73%)b 9.14 6 0.64 (105% 6 4.56%)
a Values are mean 6 standard error of the mean; n 5 12, no insulin; n 5 11, insulin during reperfusion. Epinephrine (1 mmol/L) was added in both groups for the last 20 minutes (late reperfusion). When insulin was present, it was added at the beginning of reperfusion (early b p , 0.05 compared with no insulin. reperfusion).
Results Cardiac Power Table 1 shows cardiac power before ischemia and during the early and late reperfusion periods (see Fig 1 for perfusion protocol). Cardiac power before ischemia was stable and there was no difference between the control (no insulin) and experimental groups (insulin during reperfusion). Contractile performance ceased during
Tissue Analysis The frozen tissue, ground under liquid nitrogen, was extracted with 6% perchloric acid. The tissue extracts were neutralized and immediately assayed for glucose 6-phosphate, lactate, citrate, and malate by standard enzymatic methods. Glycogen was assayed by the method of Walaas and Walaas using amyloglucosidase (described in [11]). A small portion of the pulverized tissue was dried in an oven (70°C) to constant weight and the wet-to-dry ratio was calculated.
Statistical Analysis All data are presented as mean 6 standard error of the mean. Statistical comparison was by one-way repeated measures analysis of variance (Table 1, Figs 2 and 3), accounting for time and group interaction, or by unpaired analysis of variance (metabolite data, Fig 4) with post hoc comparison by Newman-Keuls test [12]. Differences were considered statistically significant when p , 0.05.
Fig 2. Rates of glucose uptake before ischemia, during early reperfusion, and during late reperfusion in the group without insulin (control, solid bars, n 5 8) and the group with insulin during reperfusion (hatched bars, n 5 7). Glucose uptake was assessed by the cumulative release of 3H2O from [2-3H]glucose. Rates were obtained for each interval by linear regression analysis of the respective section of the slope. Note the significant increase in glucose uptake during late reperfusion. Values are mean 6 standard error of the mean. (*p , 0.01 vs before ischemia; 1p , 0.01 vs control.) Statistical comparison was by one-way repeated measures analysis of variance, accounting for time and group interaction.
Ann Thorac Surg 1999;67:1682– 8
DOENST ET AL INOTROPIC EFFECTS OF INSULIN DURING REPERFUSION
1685
Fig 4. Myocardial glycogen content in the absence (solid bars) or presence (hatched bars) of insulin. Glycogen content was determined before ischemia (n 5 4), after 15 minutes of no-flow ischemia (n 5 5), after 20 minutes of reperfusion (n 5 5 with insulin, n 5 5 without insulin), and after 20 minutes of reperfusion plus an additional 20 minutes of reperfusion in the presence of epinephrine (n 5 5 with insulin, n 5 5 without insulin). See Figure 1 for perfusion protocol. Values are mean 6 standard error of the mean. (*p , 0.01 vs before ischemia; 1p , 0.05 vs control.)
0.05). The addition of epinephrine at 55 minutes further increased recovery to 92.6% of preischemic power in the absence and to 105% in the presence of insulin.
Glucose Uptake
Fig 3. Rates of glucose oxidation (A) and oleate oxidation (B) before ischemia, during early reperfusion, and during late reperfusion in the group without insulin (control, solid bars, n 5 4) and the group with insulin during reperfusion (hatched bars, n 5 4). Glucose oxidation was assessed by the cumulative production of 14CO2 from [U-14C]glucose. Oleate oxidation was assessed by the cumulative release of 3H2O from [9,10-3H]oleate. Rates were obtained for each interval by linear regression analysis of the respective section of the slope. Note the significant increase in glucose oxidation during late reperfusion. Values are mean 6 standard error of the mean. (*p , 0.01 vs before ischemia; 1p , 0.01 vs control.) Statistical comparison was by one-way repeated measures analysis of variance, accounting for time and group interaction.
ischemia. During the first 20 minutes of reperfusion, hearts recovered 51.1% of preischemic cardiac power in the absence and 76.4% in the presence of insulin (p ,
Figure 2 shows glucose uptake of the two groups before ischemia, during early reperfusion, and during late reperfusion. There was no difference in glucose uptake between the groups during the first 20 minutes of the protocol (before ischemia). Glucose uptake during early reperfusion was not different from the initial perfusion period. The addition of epinephrine at the beginning of late reperfusion increased glucose uptake significantly in both groups. Importantly, the increase in glucose uptake was significantly greater in the presence of insulin compared with epinephrine alone. Thus, the effects of epinephrine and insulin on glucose uptake were additive.
Glucose and Oleate Oxidation Figure 3 shows the rates of glucose oxidation (upper panel) and of oleate oxidation (lower panel) in the two groups before ischemia, during early reperfusion, and during late reperfusion. Glucose oxidation was not different among groups before ischemia and during early reperfusion. Epinephrine caused a significant increase in glucose oxidation that was also potentiated in the presence of insulin. There was no difference in oleate oxidation between the two groups. Contrary to expectations [7], there were no changes in the rate of oleate oxidation during early or late reperfusion.
1686
DOENST ET AL INOTROPIC EFFECTS OF INSULIN DURING REPERFUSION
Tissue Metabolites To track changes in the major metabolite pools of carbohydrate metabolism, we determined glycogen, glucose 6-phosphate, lactate, citrate, and malate before ischemia, at the end of ischemia, after early reperfusion, and at the end of the experiments. These metabolites were chosen for their respective roles as endogenous substrates, intermediates in metabolic pathways, and regulators of key enzymes. Figure 4 shows the tissue content of glycogen at the four points mentioned above. Ischemia caused a significant reduction in myocardial glycogen content. This net glycogen breakdown was partially reversed after 20 minutes of reperfusion, both in the presence and in the absence of insulin. The addition of epinephrine resulted in renewed net glycogen breakdown. No net glycogen breakdown was observed in the presence of insulin. Except for glycogen, there were no differences between the control and the experimental groups at any of the points when metabolites were determined. The changes from one point to the other were the same in both groups. Values were as follows: Glucose-6-phosphate levels were increased after ischemia (from 0.52 6 0.14 to 1.69 6 0.21 mmol/g dry weight, p , 0.01) and remained elevated for the duration of the experiment. Tissue lactate content rose from 9.41 6 3.12 mmol/g dry weight before ischemia to 70.0 6 7.87 mmol/g dry weight at the end of ischemia ( p , 0.001) and returned to preischemic values with reperfusion. Neither insulin nor epinephrine changed tissue lactate content. We determined citrate because citrate is an allosteric regulator of the enzyme phosphofructokinase. Citrate levels were 1.64 6 0.11 mmol/g dry weight before ischemia and decreased to 0.59 6 0.14 mmol/g dry weight at the end of ischemia. Citrate content increased 4.5 to 5-fold during early reperfusion and returned to preischemic values on stimulation with epinephrine. We determined malate levels because malate is a representative citric acid cycle intermediate and a component of the malate-aspartate shuttle. Malate levels before ischemia were 0.58 6 0.06 mmol/g dry weight in the control group. During ischemia malate increased to 0.97 6 0.19 mmol/g dry weight ( p , 0.05) and then remained elevated during early reperfusion. Malate levels further increased with the addition of epinephrine ( p , 0.05) during late reperfusion (1.79 6 0.32 mmol/g dry weight without insulin, 1.97 6 0.42 mmol/g dry weight with insulin).
Comment This study shows the following: (1) Insulin has a direct positive inotropic effect on the postischemic heart. This effect was observed in the presence of high concentrations of a long-chain fatty acid and was additive to the inotropic effect of epinephrine. (2) Neither glucose nor oleate oxidation were increased during early reperfusion. Addition of epinephrine increased glucose oxidation, but not oleate oxidation. The increase in glucose oxidation was further augmented by insulin. (3) Insulin reverses
Ann Thorac Surg 1999;67:1682– 8
the glycogen depletion observed with epinephrine during late reperfusion. The results provide further evidence for the concept of metabolic support for the failing, catecholamine-stimulated, postischemic myocardium. Because of possible differences between the effects of insulin in vitro and in vivo, a brief discussion of the experimental model is in order.
Experimental Model The isolated working rat heart is an appropriate model for assessing the direct effects of insulin on the myocardium during reperfusion after total, global ischemia for the following reasons: first, the isolated working rat heart allows for the complete control of the perfusion medium under conditions in which contractile function is similar to the work performed by the heart in vivo. Second, the assessment of contractile function in terms of cardiac power is appropriate because it correlates well with oxygen consumption [8]. Third, a 15-minute period of ischemia in the isolated rat heart causes a significant degree of stunning [13]. Considerable irreversible ischemic injury occurs after 20 to 30 minutes of total normothermic ischemia in rat heart. Our experimental design was aimed to test the effects of insulin on dysfunctional but viable, ie, stunned myocardium. The use of GIK in the clinical setting is also aimed at salvaging stunned myocardium; however, the conditions that lead to mechanical dysfunction may be less defined (various durations of cardioplegic arrest during cardiopulmonary bypass, acute myocardial infarction). The assessment of contractile function in a model of ischemia-induced contractile dysfunction appears to be a plausible way to assess the effects of any intervention, eg, insulin.
Insulin and Inotropy Inotropic effects of insulin on the heart have been reported before in vivo and in vitro [14, 15]. A distinction between direct and indirect effects of insulin can only be made by comparing in vivo studies with studies in the isolated heart. In vivo studies showed that insulin causes vasodilation in skeletal muscle [16], thus decreasing afterload, and that insulin shifts metabolism of the heart from fatty acid oxidation to glucose oxidation [17]. In addition, high levels of insulin give rise to an increased adrenergic tone [18]. All of these observations would provide a plausible explanation for the inotropic effects of insulin. We now demonstrate in the isolated working heart a direct inotropic effect, which precedes the changes in glucose uptake and oxidation. This finding is important because it suggests that there is an insulinmediated pathway in the heart that increases contractile function independent of the effects of insulin on glucose metabolism. The present findings are also important because they were obtained in the presence of physiologic concentrations of glucose and fatty acids. Although the temporal dissociation between the inotropic and the metabolic effects of insulin argues against a direct causal relationship, it is reasonable to assume that the two effects are linked, because increased function requires increased energy production. These results also
1687
Ann Thorac Surg 1999;67:1682– 8
DOENST ET AL INOTROPIC EFFECTS OF INSULIN DURING REPERFUSION
suggest that the inotropic and the metabolic effects of insulin are mediated through different insulin-activated pathways. These pathways require further elucidation.
breakdown, which is reversible during early reperfusion (Fig 4). Epinephrine, when added at the beginning of late reperfusion, prevents net glycogen resynthesis. The epinephrine effect is reversed by insulin, suggesting that insulin increases rates of glycogen synthesis rather than decreasing rates of glycogen breakdown. This interpretation is consistent with our previous observation of enhanced rates of glycogen synthesis in the reperfused working rat heart after low-flow ischemia [23]. We have shown earlier that glycogen depletion before ischemia worsens the return of contractile function after ischemia [24] and that “glycogen loading” before ischemia improves ischemia tolerance [11, 13, 25]. It is reasonable to assume that glycogen is more than an “energy buffer” for the stressed heart. Recent observations in mouse skeletal muscle show that reductions in force, Ca21 release, and contractile protein inhibition are closely associated with reduced muscle glycogen concentrations [26]. It is tempting to speculate that a similar association exists in heart muscle, although the present system did not allow us to test this hypothesis.
Possible Mechanisms Postischemic glucose metabolism may be beneficial for recovery of function during reperfusion, whereas the reliance on fatty acids as energy substrate may be detrimental [19]. Insulin causes a shift from utilization of fatty acids to glucose [17]. Several reasons have been suggested to explain the beneficial effects of glucose utilization. First, utilizing glucose maintains basal adenosine triphosphate production through anaerobic glycolysis. Second, glucose serves as an anaplerotic fuel to replenish metabolite pools that have been depleted during ischemia [8]. Third, the oxidation of carbohydrate as substrate is more efficient than fatty acids. Fourth, the oxidation of fatty acids in the absence of sufficient oxygen may lead to the accumulation of toxic intermediates, eg, acyl-carnitine. Acyl-carnitine inhibits the Ca21 pump, the Na1-Ca21 exchanger, and the Na1-K1 pump of the sarcolemma. It also activates Ca21 channels [20]. These events may cause an intracellular Ca21 overload. In this study, glucose oxidation was increased during reperfusion but oleate oxidation did not change (Fig 3). These results are at odds with those of Kudo and associates [7], who found an increase in fatty acid oxidation during reperfusion after 30 minutes of total ischemia in the same experimental model. The authors suggested that the increase in fatty acid oxidation during reperfusion is caused by the activation of 59 adenosine monophosphate–activated protein kinase by adenosine monophosphate accumulation during ischemia. Adenosine monophosphate–activated protein kinase phosphorylates and inactivates acetyl CoA carboxylase. Inhibition of acetyl CoA carboxylase leads to a decrease in malonyl CoA, a potent inhibitor of carnitine palmitoyl transferase I (CPT-I), the primary regulatory step for fatty acid oxidation. The same group advanced a similar hypothesis for the insulin-induced decrease in fatty acid oxidation during normoxic perfusion [21]. These investigators suggested that insulin inhibited adenosine monophosphate– activated protein kinase, thus decreasing fatty acid oxidation. This hypothesis is also not supported by the present results obtained in the presence of insulin during reperfusion, because fatty acid oxidation remained unchanged in the presence of insulin. From the present results, it is reasonable to conclude that the postischemic heart is using more energy-providing substrates per unit of work during the recovery of function than during the preischemic period. This conclusion supports the notion that glucose metabolism during reperfusion is not primarily used to generate adenosine triphosphate for contraction but may be directed to restoration of glycogen and replenishment of depleted intermediates. A glucose requirement for postischemic recovery of perfused working hearts is well established [22]. The importance of glycogen for the postischemic recovery of function in isolated working rat heart is also well established [11]. Ischemia results in net glycogen
Conclusions Insulin has a direct positive inotropic effect on postischemic rat heart. This effect is additive to epinephrine. Whereas the positive inotropic effect of insulin is immediate, increased rates of glucose oxidation and net glycogen synthesis occur after a delay. The results support the notion that insulin may augment the inotropic effects of epinephrine on the heart in vivo.
Clinical Implications Glucose, insulin, and potassium solution is used clinically to treat postischemic contractile dysfunction in cardiogenic shock after aortocoronary bypass graft surgery [1, 19] and to treat patients with acute myocardial infarction [5]. Pooled data from trials of high-dose GIK in acute myocardial infarction showed an association between reduction of free fatty acid levels (because of the antilipolytic effects of insulin) and reduction in mortality. It has been speculated that the combination of reperfusion, GIK, and b-blockers may have the greatest impact on reducing cardiac mortality after an acute myocardial infarction [5]. All three interventions raise myocardial glycogen levels. In light of our present findings, which show a direct inotropic effect of insulin on the postischemic myocardium, it is reasonable to assume that the beneficial effects of GIK are not caused by the reduction of free fatty acid levels alone. Our results also lend strength to the concept that insulin reduces cathecholamine requirements during early reperfusion of previously ischemic, stunned myocardium.
The work was supported in part by National Heart, Lung, and Blood Institute Grant RO1 HL-43133. T. Doenst was the recipient of a research fellowship from the German Research Foundation (Deutsche Forschungsgemeinschaft).
1688
DOENST ET AL INOTROPIC EFFECTS OF INSULIN DURING REPERFUSION
References 1. Gradinak S, Coleman GM, Taegtmeyer H, Sweeney MS, Frazier OH. Improved cardiac function with glucose-insulinpotassium after coronary bypass surgery. Ann Thorac Surg 1989;48:484–9. 2. Sodi-Pallares D, Testelli MR, Fishleder BL, et al. Effects of intravenous infusion of a potassium-glucose-insulin solution on the electrocardiographic signs of myocardial infarction. Am J Cardiol 1962;9:166– 81. 3. Neely JR, Grotyohann LW. Role of glycolytic products in damage to myocardium: dissociation of adenosine triphosphate levels and recovery of function of reperfused canine myocardium. Circ Res 1984;55:816–24. 4. Apstein CS, Gravino FN, Haudenschild CC. Determinants of a protective effect of glucose and insulin on the ischemic myocardium. Effects on contractile function, diastolic compliance, metabolism, and ultrastructure during ischemia and reperfusion. Circ Res 1983;52:515–26. 5. Fath-Ordoubadi F, Beatt KJ. Glucose-insulin-potassium therapy for treatment of acute myocardial infarction. An overview of randomized placebo-controlled trials. Circulation 1997;96:1152– 6. 6. Taegtmeyer H, Roberts AFC, Rayne AEG. Energy metabolism in reperfused rat heart: return of function before normalization of ATP content. J Am Coll Cardiol 1985;6: 864–70. 7. Kudo N, Barr AJ, Barr RL, Desai S, Lopaschuk GD. High rates of fatty acid oxidation during reperfusion of ischemic hearts are associated with a decrease in malonyl-CoA levels due to an increase in 59-AMP-activated protein kinase inhibition of acetyl-Coa carboxylase. J Biol Chem 1995;270: 17513–20. 8. Goodwin GW, Taegtmeyer H. Metabolic recovery of the isolated working rat heart after brief global ischemia. Am J Physiol 1994;267:H462–70. 9. Taegtmeyer H, Hems R, Krebs HA. Utilization of energy providing substrates in the isolated working rat heart. Biochem J 1980;186:701–11. 10. Goodwin GW, Arteaga JA, Taegtmeyer H. Glycogen turnover in the isolated working rat heart. J Biol Chem 1995;270: 9234– 40. 11. Doenst T, Guthrie PH, Chemnitius J-M, Zech R, Taegtmeyer H. Fasting, lactate, and insulin improve ischemia tolerance: a comparison with ischemic preconditioning. Am J Physiol 1996;270:H1607–15. 12. Zar J. Biostatistical analysis, 2nd ed. Inglewood Cliffs: Prentice-Hall, 1984.
Ann Thorac Surg 1999;67:1682– 8
13. Schneider CA, Taegtmeyer H. Fasting in vivo delays myocardial cell damage after brief periods of ischemia in the isolated working rat heart. Circ Res 1991;68:1045–50. 14. Depre C, Hue L. Inhibition of glyocogenolysis by a glucose analogue in the working rat heart. J Mol Cell Cardiol 1997;29: 2253–9. 15. Ferranini E, Santoro D, Bonadonnar R, Natali A, Parodi O, Camici P. Metabolic and hemodynamic effects of insulin on human hearts. Am J Physiol 1993;264:E308 –15. 16. Baron AD. Hemodynamic actions of insulin. Am J Physiol 1994;267:E187–222. 17. Barrett E, Schwartz R, Francis C, Zaret B. Regulation by insulin of myocardial glucose and fatty acid metabolism in the conscious dog. J Clin Invest 1984;74:1073–9. 18. Scherrer U, Sartori C. Insulin as a vascular and sympathoexcitatory hormone. Implications for blood pressure regulation, insulin sensitivity, and cardiovascular morbidity. Circulation 1997;96:4104–13. 19. Taegtmeyer H, Goodwin GW, Doenst T, Frazier OH. Substrate metabolism as a determinant for postischemic functional recovery of the heart. Am J Cardiol 1997;80:3A–10A. 20. Hendrickson SC, St. Louis JD, Lowe JE, Abdel-aleem S. Free fatty acid metabolism during myocardial ischemia and reperfusion. Mol Cell Biochem 1997;166:85–94. 21. Gamble J, Lopaschuk GD. Insulin inhibition of 59 adenosine monophosphate–activated protein kinase in the heart results in activation of acetyl coenzyme A carboxylase and inhibition of fatty acid metabolism. Metabolism 1997;46:1270– 4. 22. Mallet RT, Hartman DA, Bu¨nger R. Glucose requirement for postischemic recovery of perfused working heart. Eur J Biochem 1990;188:481–93. 23. Bolukoglu H, Goodwin GW, Guthrie PH, Carmical SG, Chen TM, Taegtmeyer H. Metabolic fate of glucose in reversible low-flow ischemia of the isolated working rat heart. Am J Physiol 1996;270:H817–26. 24. Lagerstrom CF, Walker WE, Taegtmeyer H. Failure of glycogen depletion to improve left ventricular function of the rabbit heart after hypothermic ischemic arrest. Circ Res 1988;63:81– 6. 25. McElroy DD, Walker WE, Taegtmeyer H. Glycogen loading improves left ventricular function of the rabbit heart after hypothermic ischemic arrest. J Appl Cardiol 1989;4:455– 65. 26. Chin E, Allen D. Effects of reduced muscle glycogen concentration on force, Ca21 release and contractile protein function in intact mouse skeletal muscle. J Physiol (Lond) 1997; 498:17–29.
Background. Glucose, insulin, and potassium solution improves left ventricular function in refractory pump failure. Direct effects of insulin on the heart cannot be determined in vivo. We hypothesized that insulin has a direct positive inotropic effect on the reperfused heart. Methods. Isolated working rat hearts were perfused with buffer containing glucose (5 mmol/L) plus oleate (1.2 mmol/L). Hearts were subjected to 15 minutes of ischemia and reperfused with or without insulin (100 mU/mL) for 40 minutes. Epinephrine (1 mmol/L) was added for the last 20 minutes. Results. Hearts recovered 51.1% of preischemic cardiac power in the absence and 76.4% in the presence of insulin (p < 0.05). Whereas oleate oxidation remained
unchanged, glucose uptake and oxidation increased during reperfusion with epinephrine (p < 0.01). This increase was significantly greater when hearts were reperfused in the presence of insulin (p < 0.01). Insulin also prevented an epinephrine-induced glycogen breakdown during reperfusion (p < 0.05). Conclusions. Insulin has a direct positive inotropic effect on postischemic rat heart. This effect is additive to epinephrine and occurs without delay. Increased rates of glucose oxidation and net glycogen synthesis are more protracted.
T
tion of the ischemic and reperfused myocardium are not known. It is not even certain whether insulin and enhanced glucose substrate metabolism affect the contractile function of the heart. The cardiac effects of GIK could be mediated by the systemic effects of insulin, which lower free fatty acid levels in the plasma and decrease systemic vascular resistance. With the advent of new methods of myocardial preservation and reperfusion of previously ischemic myocardium, the concept of substrate selection and metabolic support for the reperfused myocardium deserves to be reexamined in light of newer concepts of energy transfer in heart muscle. Ischemia is characterized by the depletion of glycogen and citric acid cycle intermediates [6]. Reperfusion metabolism is characterized by high rates of long-chain fatty acid oxidation, impaired glucose oxidation, and the uncoupling of energy production and utilization [7]. Even when glucose is the only substrate added to the perfusate, glucose utilization is depressed during reperfusion in the isolated working rat heart [8]. Regardless of the specific mechanism, there is reasonable support for the hypothesis that myocardial substrate metabolism per se has a direct effect on the contractile function of the postischemic, reperfused heart, which is mediated by enhanced rates of glucose oxidation. Glucose uptake and oxidation are, however, inhibited by long-chain fatty acids. Insulin may overcome the inhibition of glucose metabolism by mechanisms not yet completely understood. We tested the hypothesis that insulin has a direct positive inotropic effect on the reperfused heart and that this effect is mediated by increased rates of
he present study was undertaken to test the efficacy of insulin for recovery of function and glucose metabolism in the postischemic heart. The starting point was our observation that infusion of glucose, insulin, and potassium (GIK) solution improves contractile performance and reduces morbidity in patients with refractory heart failure after cardiopulmonary bypass [1]. Although the concept of metabolic support for the ischemic heart reperfused with GIK is not new, the rationale for the “polarizing solution” [2] was that insulin stimulates K1 reuptake and reduces the development of arrhythmias. However, some investigators have pointed out that enhanced rates of glycolysis result in H1 accumulation and impair contractile function during reperfusion [3], whereas others have observed that glycolysis preserves energy flux and contractile function of the heart during ischemia and reperfusion [4]. Clinical trials with GIK have shown a significant reduction in mortality of patients treated with GIK either in the course of an acute myocardial infarction [5] or in the course of refractory left ventricular failure after hypothermic ischemic arrest for aortocoronary bypass graft surgery [1]. Although the clinical implications of improved energy substrate metabolism seem obvious, the mechanisms by which alterations of metabolism can benefit the contracAccepted for publication Dec 16, 1998. Address reprint requests to Dr Taegtmeyer, Division of Cardiology, Department of Internal Medicine, University of Texas-Houston Medical School, 6431 Fannin, 1.246 MSB, Houston, TX 77030; e-mail: [email protected].
© 1999 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
(Ann Thorac Surg 1999;67:1682– 8) © 1999 by The Society of Thoracic Surgeons
0003-4975/99/$20.00 PII S0003-4975(99)00326-4
Ann Thorac Surg 1999;67:1682– 8
DOENST ET AL INOTROPIC EFFECTS OF INSULIN DURING REPERFUSION
1683
glucose oxidation, increased myocardial glycogen levels, and replenishment (anaplerosis) of the citric acid cycle. We used the isolated working rat heart perfused with physiologic concentrations of glucose and oleate, subjected it to total, global ischemia, and exposed it to epinephrine during reperfusion to mimic the clinical situation of reperfusion after controlled ischemic arrest of the heart.
Material and Methods Animals Male Sprague-Dawley rats (300 to 400 g) were obtained from Harlan (Indianapolis, IN) and housed in the University of Texas-Houston Medical School Animal Care Center. All animals had free access to food and water until the time of the experiment. All experiments were performed under protocols approved by The University of Texas-Houston Medical School. Animals received humane care in compliance with the “Guide for the Care and Use of Laboratory Animals” (NIH publication 85-23, revised 1985).
Materials d-[U-14C]glucose was obtained from ICN Biochemicals (Costa Mesa, CA). d-[2-3H]glucose was from Amersham Corp (Arlington Height, IL). [9,10-3H]oleate was from Sigma Chemical Co (St. Louis, MO). The purity of the 3 H-labeled tracers were ascertained by measuring the intrinsic 3H2O content. Fatty acid-free bovine serum albumin was obtained from Intergen Co (Purchase, NY). Enzymes for metabolite assays were from Boehringer Mannheim (Indianapolis, IN). All other chemicals were purchased from Sigma.
Working Heart Preparation The preparation has been described in detail earlier [9]. The perfusion apparatus has been modified to allow the collection of 14CO2 [10]. The apparatus was rendered gas tight and the exhaust was bubbled through a solution of 0.33 mmol/L benzethonium hydroxide for the collection of 14CO2 in the gaseous phase. Hearts were perfused as working hearts at 37°C with recirculating KrebsHenseleit buffer (200 mL) containing glucose (5 mmol/L) plus sodium-oleate (1.2 mmol/L) bound to 3% bovine serum albumin, Cohn fraction V, fatty acid-free, and equilibrated with 95% O2–5% CO2. Total perfusate [Ca21] was 2.5 mmol/L. All experiments were carried out with a preload of 15 cm H2O and an afterload of 100 cm H2O. The hearts were beating spontaneously. Aortic flow and coronary flow were measured every 5 minutes. Heart rate as well as systolic and diastolic aortic pressures were measured continuously with a 3F Millar transducer (Millar Instruments, Houston, TX) and a MacLab physiologic recording system (ADInstruments, Milford, MA).
Perfusion Protocol The perfusion protocol is shown in Figure 1. Hearts were perfused for 20 minutes under baseline conditions. Total,
Fig 1. Schematic of the experimental protocol and substrates used. Isolated working rat hearts were perfused with Krebs-Henseleit buffer at a preload of 15 cm H2O and an afterload of 100 cm H2O. See Material and Methods for details.
global, normothermic ischemia was induced for 15 minutes by clamping, and reperfusion was induced by unclamping, both the aortic and the atrial line of the perfusion system. In the experimental group, insulin (100 mU/mL) was added at the beginning of reperfusion (n 5 11, n 5 7 with [2-3H]glucose as tracer, n 5 4 with [U-14C]glucose and [9,10-3H]oleate as tracers). The control group (n 5 12, n 5 8 with [2-3H]glucose as tracer, n 5 4 with [U-14C]glucose and [9,10-3H]oleate as tracers) was reperfused without insulin. In both groups epinephrine (1 mmmol/L) was added after 20 minutes of reperfusion. Hearts were freeze-clamped 20 minutes after the addition of epinephrine. The use of the different tracer protocols did not affect contractile function or glycogen content of the hearts. In separate experiments, hearts were perfused according to the same protocol but were freeze-clamped before ischemia (n 5 4), at the end of ischemia (n 5 4), or after 20 minutes of reperfusion (n 5 5 with insulin, n 5 5 without insulin) for the determination of metabolites. Cardiac performance of these hearts was not different from those perfused for the entire duration of the experimental protocol (data not shown).
Assessment of Contractile Performance Cardiac performance was expressed as cardiac power (the product of cardiac output and mean aortic pressure) in terms of milliwatts as described earlier [11]. Mean aortic pressure (cm H2O) was calculated as (systolic 1 diastolic pressure 3 2)/3. Heart rate was measured as beats per minute and cardiac output as milliliters per minute. We used the average value of cardiac power during the last 10 minutes of the preischemic period as the reference value (baseline) for postischemic cardiac performance. Recovery of power during reperfusion was assessed separately for the time period before and after the addition of epinephrine. Recovery was determined as the mean value of all cardiac power measurements
1684
DOENST ET AL INOTROPIC EFFECTS OF INSULIN DURING REPERFUSION
during the first 20 minutes of reperfusion (early reperfusion) and during the last 20 minutes of reperfusion.
Ann Thorac Surg 1999;67:1682– 8
Table 1. Cardiac Power Before Ischemia, During Early Reperfusion, and Late Reperfusion With Epinephrinea Cardiac Power (mW)
Assessment of Uptake and Oxidation Rates Dual-label counting of 3H and 14C was performed on a Packard 1900 TR liquid scintillation analyzer by the method of spectral index analysis as described by the manufacturer (Packard Instruments, Meriden, CT). Glucose uptake and oleate oxidation were determined by the rate of 3H2O production from [2-3H]glucose and [9,10-3H]oleate, respectively [10, 11]. Release of 3H2O into the perfusate was analyzed in 5-minute intervals. 3H2O was separated from [2-3H]glucose or [9,10-3H]oleate in the perfusate by anion exchange chromatography on AG-1X8 resin (BioRad Laboratories, Hercules, CA) [10]. The amount of 3H2O in the perfusate was plotted against time, and the slopes of the desired intervals were used to calculate glucose uptake or oleate oxidation rates, which were expressed as micromoles per minute per gram of dry weight. Glucose oxidation was determined from the cumulative production of 14CO2 from [U-14C]glucose. The amount of 14CO2 present in both the exhaust and the perfusate was assessed. The exhausted 14CO2 was collected during 5-minute intervals by bubbling through 5 mL of 0.33 mmol/L benzethonium hydroxide in methanol. The content of 14CO2 in the perfusate was determined by trapping 14CO2 liberated on acidifying 500 mL of the perfusate samples in 1 mL of 1 mmol/L benzethonium hydroxide in methanol. Quantitative recovery of 14 CO2 was verified in a preliminary experiment by injecting a bolus of [14C]NaHCO3 (2 mCi) into the sealed perfusion apparatus and calculating recovery after determining 14CO2 in exhaust and perfusate. The sum of 14 CO2 in the perfusate and the exhaust was plotted against time, and the slopes of the desired intervals were used to calculate glucose oxidation rates, which were expressed as micromoles per minute per gram of dry weight.
Observation Period Before ischemia (0 –20 min) Early reperfusion (35–55 min) Late reperfusion (55–75 min)
No Insulin
Insulin During Reperfusion
8.54 6 0.90 (100%) 4.13 6 0.42 (51.1% 6 5.18%) 7.49 6 0.64 (92.6% 6 10.6%)
8.76 6 0.54 (100%) 6.89 6 0.78b (76.4% 6 4.73%)b 9.14 6 0.64 (105% 6 4.56%)
a Values are mean 6 standard error of the mean; n 5 12, no insulin; n 5 11, insulin during reperfusion. Epinephrine (1 mmol/L) was added in both groups for the last 20 minutes (late reperfusion). When insulin was present, it was added at the beginning of reperfusion (early b p , 0.05 compared with no insulin. reperfusion).
Results Cardiac Power Table 1 shows cardiac power before ischemia and during the early and late reperfusion periods (see Fig 1 for perfusion protocol). Cardiac power before ischemia was stable and there was no difference between the control (no insulin) and experimental groups (insulin during reperfusion). Contractile performance ceased during
Tissue Analysis The frozen tissue, ground under liquid nitrogen, was extracted with 6% perchloric acid. The tissue extracts were neutralized and immediately assayed for glucose 6-phosphate, lactate, citrate, and malate by standard enzymatic methods. Glycogen was assayed by the method of Walaas and Walaas using amyloglucosidase (described in [11]). A small portion of the pulverized tissue was dried in an oven (70°C) to constant weight and the wet-to-dry ratio was calculated.
Statistical Analysis All data are presented as mean 6 standard error of the mean. Statistical comparison was by one-way repeated measures analysis of variance (Table 1, Figs 2 and 3), accounting for time and group interaction, or by unpaired analysis of variance (metabolite data, Fig 4) with post hoc comparison by Newman-Keuls test [12]. Differences were considered statistically significant when p , 0.05.
Fig 2. Rates of glucose uptake before ischemia, during early reperfusion, and during late reperfusion in the group without insulin (control, solid bars, n 5 8) and the group with insulin during reperfusion (hatched bars, n 5 7). Glucose uptake was assessed by the cumulative release of 3H2O from [2-3H]glucose. Rates were obtained for each interval by linear regression analysis of the respective section of the slope. Note the significant increase in glucose uptake during late reperfusion. Values are mean 6 standard error of the mean. (*p , 0.01 vs before ischemia; 1p , 0.01 vs control.) Statistical comparison was by one-way repeated measures analysis of variance, accounting for time and group interaction.
Ann Thorac Surg 1999;67:1682– 8
DOENST ET AL INOTROPIC EFFECTS OF INSULIN DURING REPERFUSION
1685
Fig 4. Myocardial glycogen content in the absence (solid bars) or presence (hatched bars) of insulin. Glycogen content was determined before ischemia (n 5 4), after 15 minutes of no-flow ischemia (n 5 5), after 20 minutes of reperfusion (n 5 5 with insulin, n 5 5 without insulin), and after 20 minutes of reperfusion plus an additional 20 minutes of reperfusion in the presence of epinephrine (n 5 5 with insulin, n 5 5 without insulin). See Figure 1 for perfusion protocol. Values are mean 6 standard error of the mean. (*p , 0.01 vs before ischemia; 1p , 0.05 vs control.)
0.05). The addition of epinephrine at 55 minutes further increased recovery to 92.6% of preischemic power in the absence and to 105% in the presence of insulin.
Glucose Uptake
Fig 3. Rates of glucose oxidation (A) and oleate oxidation (B) before ischemia, during early reperfusion, and during late reperfusion in the group without insulin (control, solid bars, n 5 4) and the group with insulin during reperfusion (hatched bars, n 5 4). Glucose oxidation was assessed by the cumulative production of 14CO2 from [U-14C]glucose. Oleate oxidation was assessed by the cumulative release of 3H2O from [9,10-3H]oleate. Rates were obtained for each interval by linear regression analysis of the respective section of the slope. Note the significant increase in glucose oxidation during late reperfusion. Values are mean 6 standard error of the mean. (*p , 0.01 vs before ischemia; 1p , 0.01 vs control.) Statistical comparison was by one-way repeated measures analysis of variance, accounting for time and group interaction.
ischemia. During the first 20 minutes of reperfusion, hearts recovered 51.1% of preischemic cardiac power in the absence and 76.4% in the presence of insulin (p ,
Figure 2 shows glucose uptake of the two groups before ischemia, during early reperfusion, and during late reperfusion. There was no difference in glucose uptake between the groups during the first 20 minutes of the protocol (before ischemia). Glucose uptake during early reperfusion was not different from the initial perfusion period. The addition of epinephrine at the beginning of late reperfusion increased glucose uptake significantly in both groups. Importantly, the increase in glucose uptake was significantly greater in the presence of insulin compared with epinephrine alone. Thus, the effects of epinephrine and insulin on glucose uptake were additive.
Glucose and Oleate Oxidation Figure 3 shows the rates of glucose oxidation (upper panel) and of oleate oxidation (lower panel) in the two groups before ischemia, during early reperfusion, and during late reperfusion. Glucose oxidation was not different among groups before ischemia and during early reperfusion. Epinephrine caused a significant increase in glucose oxidation that was also potentiated in the presence of insulin. There was no difference in oleate oxidation between the two groups. Contrary to expectations [7], there were no changes in the rate of oleate oxidation during early or late reperfusion.
1686
DOENST ET AL INOTROPIC EFFECTS OF INSULIN DURING REPERFUSION
Tissue Metabolites To track changes in the major metabolite pools of carbohydrate metabolism, we determined glycogen, glucose 6-phosphate, lactate, citrate, and malate before ischemia, at the end of ischemia, after early reperfusion, and at the end of the experiments. These metabolites were chosen for their respective roles as endogenous substrates, intermediates in metabolic pathways, and regulators of key enzymes. Figure 4 shows the tissue content of glycogen at the four points mentioned above. Ischemia caused a significant reduction in myocardial glycogen content. This net glycogen breakdown was partially reversed after 20 minutes of reperfusion, both in the presence and in the absence of insulin. The addition of epinephrine resulted in renewed net glycogen breakdown. No net glycogen breakdown was observed in the presence of insulin. Except for glycogen, there were no differences between the control and the experimental groups at any of the points when metabolites were determined. The changes from one point to the other were the same in both groups. Values were as follows: Glucose-6-phosphate levels were increased after ischemia (from 0.52 6 0.14 to 1.69 6 0.21 mmol/g dry weight, p , 0.01) and remained elevated for the duration of the experiment. Tissue lactate content rose from 9.41 6 3.12 mmol/g dry weight before ischemia to 70.0 6 7.87 mmol/g dry weight at the end of ischemia ( p , 0.001) and returned to preischemic values with reperfusion. Neither insulin nor epinephrine changed tissue lactate content. We determined citrate because citrate is an allosteric regulator of the enzyme phosphofructokinase. Citrate levels were 1.64 6 0.11 mmol/g dry weight before ischemia and decreased to 0.59 6 0.14 mmol/g dry weight at the end of ischemia. Citrate content increased 4.5 to 5-fold during early reperfusion and returned to preischemic values on stimulation with epinephrine. We determined malate levels because malate is a representative citric acid cycle intermediate and a component of the malate-aspartate shuttle. Malate levels before ischemia were 0.58 6 0.06 mmol/g dry weight in the control group. During ischemia malate increased to 0.97 6 0.19 mmol/g dry weight ( p , 0.05) and then remained elevated during early reperfusion. Malate levels further increased with the addition of epinephrine ( p , 0.05) during late reperfusion (1.79 6 0.32 mmol/g dry weight without insulin, 1.97 6 0.42 mmol/g dry weight with insulin).
Comment This study shows the following: (1) Insulin has a direct positive inotropic effect on the postischemic heart. This effect was observed in the presence of high concentrations of a long-chain fatty acid and was additive to the inotropic effect of epinephrine. (2) Neither glucose nor oleate oxidation were increased during early reperfusion. Addition of epinephrine increased glucose oxidation, but not oleate oxidation. The increase in glucose oxidation was further augmented by insulin. (3) Insulin reverses
Ann Thorac Surg 1999;67:1682– 8
the glycogen depletion observed with epinephrine during late reperfusion. The results provide further evidence for the concept of metabolic support for the failing, catecholamine-stimulated, postischemic myocardium. Because of possible differences between the effects of insulin in vitro and in vivo, a brief discussion of the experimental model is in order.
Experimental Model The isolated working rat heart is an appropriate model for assessing the direct effects of insulin on the myocardium during reperfusion after total, global ischemia for the following reasons: first, the isolated working rat heart allows for the complete control of the perfusion medium under conditions in which contractile function is similar to the work performed by the heart in vivo. Second, the assessment of contractile function in terms of cardiac power is appropriate because it correlates well with oxygen consumption [8]. Third, a 15-minute period of ischemia in the isolated rat heart causes a significant degree of stunning [13]. Considerable irreversible ischemic injury occurs after 20 to 30 minutes of total normothermic ischemia in rat heart. Our experimental design was aimed to test the effects of insulin on dysfunctional but viable, ie, stunned myocardium. The use of GIK in the clinical setting is also aimed at salvaging stunned myocardium; however, the conditions that lead to mechanical dysfunction may be less defined (various durations of cardioplegic arrest during cardiopulmonary bypass, acute myocardial infarction). The assessment of contractile function in a model of ischemia-induced contractile dysfunction appears to be a plausible way to assess the effects of any intervention, eg, insulin.
Insulin and Inotropy Inotropic effects of insulin on the heart have been reported before in vivo and in vitro [14, 15]. A distinction between direct and indirect effects of insulin can only be made by comparing in vivo studies with studies in the isolated heart. In vivo studies showed that insulin causes vasodilation in skeletal muscle [16], thus decreasing afterload, and that insulin shifts metabolism of the heart from fatty acid oxidation to glucose oxidation [17]. In addition, high levels of insulin give rise to an increased adrenergic tone [18]. All of these observations would provide a plausible explanation for the inotropic effects of insulin. We now demonstrate in the isolated working heart a direct inotropic effect, which precedes the changes in glucose uptake and oxidation. This finding is important because it suggests that there is an insulinmediated pathway in the heart that increases contractile function independent of the effects of insulin on glucose metabolism. The present findings are also important because they were obtained in the presence of physiologic concentrations of glucose and fatty acids. Although the temporal dissociation between the inotropic and the metabolic effects of insulin argues against a direct causal relationship, it is reasonable to assume that the two effects are linked, because increased function requires increased energy production. These results also
1687
Ann Thorac Surg 1999;67:1682– 8
DOENST ET AL INOTROPIC EFFECTS OF INSULIN DURING REPERFUSION
suggest that the inotropic and the metabolic effects of insulin are mediated through different insulin-activated pathways. These pathways require further elucidation.
breakdown, which is reversible during early reperfusion (Fig 4). Epinephrine, when added at the beginning of late reperfusion, prevents net glycogen resynthesis. The epinephrine effect is reversed by insulin, suggesting that insulin increases rates of glycogen synthesis rather than decreasing rates of glycogen breakdown. This interpretation is consistent with our previous observation of enhanced rates of glycogen synthesis in the reperfused working rat heart after low-flow ischemia [23]. We have shown earlier that glycogen depletion before ischemia worsens the return of contractile function after ischemia [24] and that “glycogen loading” before ischemia improves ischemia tolerance [11, 13, 25]. It is reasonable to assume that glycogen is more than an “energy buffer” for the stressed heart. Recent observations in mouse skeletal muscle show that reductions in force, Ca21 release, and contractile protein inhibition are closely associated with reduced muscle glycogen concentrations [26]. It is tempting to speculate that a similar association exists in heart muscle, although the present system did not allow us to test this hypothesis.
Possible Mechanisms Postischemic glucose metabolism may be beneficial for recovery of function during reperfusion, whereas the reliance on fatty acids as energy substrate may be detrimental [19]. Insulin causes a shift from utilization of fatty acids to glucose [17]. Several reasons have been suggested to explain the beneficial effects of glucose utilization. First, utilizing glucose maintains basal adenosine triphosphate production through anaerobic glycolysis. Second, glucose serves as an anaplerotic fuel to replenish metabolite pools that have been depleted during ischemia [8]. Third, the oxidation of carbohydrate as substrate is more efficient than fatty acids. Fourth, the oxidation of fatty acids in the absence of sufficient oxygen may lead to the accumulation of toxic intermediates, eg, acyl-carnitine. Acyl-carnitine inhibits the Ca21 pump, the Na1-Ca21 exchanger, and the Na1-K1 pump of the sarcolemma. It also activates Ca21 channels [20]. These events may cause an intracellular Ca21 overload. In this study, glucose oxidation was increased during reperfusion but oleate oxidation did not change (Fig 3). These results are at odds with those of Kudo and associates [7], who found an increase in fatty acid oxidation during reperfusion after 30 minutes of total ischemia in the same experimental model. The authors suggested that the increase in fatty acid oxidation during reperfusion is caused by the activation of 59 adenosine monophosphate–activated protein kinase by adenosine monophosphate accumulation during ischemia. Adenosine monophosphate–activated protein kinase phosphorylates and inactivates acetyl CoA carboxylase. Inhibition of acetyl CoA carboxylase leads to a decrease in malonyl CoA, a potent inhibitor of carnitine palmitoyl transferase I (CPT-I), the primary regulatory step for fatty acid oxidation. The same group advanced a similar hypothesis for the insulin-induced decrease in fatty acid oxidation during normoxic perfusion [21]. These investigators suggested that insulin inhibited adenosine monophosphate– activated protein kinase, thus decreasing fatty acid oxidation. This hypothesis is also not supported by the present results obtained in the presence of insulin during reperfusion, because fatty acid oxidation remained unchanged in the presence of insulin. From the present results, it is reasonable to conclude that the postischemic heart is using more energy-providing substrates per unit of work during the recovery of function than during the preischemic period. This conclusion supports the notion that glucose metabolism during reperfusion is not primarily used to generate adenosine triphosphate for contraction but may be directed to restoration of glycogen and replenishment of depleted intermediates. A glucose requirement for postischemic recovery of perfused working hearts is well established [22]. The importance of glycogen for the postischemic recovery of function in isolated working rat heart is also well established [11]. Ischemia results in net glycogen
Conclusions Insulin has a direct positive inotropic effect on postischemic rat heart. This effect is additive to epinephrine. Whereas the positive inotropic effect of insulin is immediate, increased rates of glucose oxidation and net glycogen synthesis occur after a delay. The results support the notion that insulin may augment the inotropic effects of epinephrine on the heart in vivo.
Clinical Implications Glucose, insulin, and potassium solution is used clinically to treat postischemic contractile dysfunction in cardiogenic shock after aortocoronary bypass graft surgery [1, 19] and to treat patients with acute myocardial infarction [5]. Pooled data from trials of high-dose GIK in acute myocardial infarction showed an association between reduction of free fatty acid levels (because of the antilipolytic effects of insulin) and reduction in mortality. It has been speculated that the combination of reperfusion, GIK, and b-blockers may have the greatest impact on reducing cardiac mortality after an acute myocardial infarction [5]. All three interventions raise myocardial glycogen levels. In light of our present findings, which show a direct inotropic effect of insulin on the postischemic myocardium, it is reasonable to assume that the beneficial effects of GIK are not caused by the reduction of free fatty acid levels alone. Our results also lend strength to the concept that insulin reduces cathecholamine requirements during early reperfusion of previously ischemic, stunned myocardium.
The work was supported in part by National Heart, Lung, and Blood Institute Grant RO1 HL-43133. T. Doenst was the recipient of a research fellowship from the German Research Foundation (Deutsche Forschungsgemeinschaft).
1688
DOENST ET AL INOTROPIC EFFECTS OF INSULIN DURING REPERFUSION
References 1. Gradinak S, Coleman GM, Taegtmeyer H, Sweeney MS, Frazier OH. Improved cardiac function with glucose-insulinpotassium after coronary bypass surgery. Ann Thorac Surg 1989;48:484–9. 2. Sodi-Pallares D, Testelli MR, Fishleder BL, et al. Effects of intravenous infusion of a potassium-glucose-insulin solution on the electrocardiographic signs of myocardial infarction. Am J Cardiol 1962;9:166– 81. 3. Neely JR, Grotyohann LW. Role of glycolytic products in damage to myocardium: dissociation of adenosine triphosphate levels and recovery of function of reperfused canine myocardium. Circ Res 1984;55:816–24. 4. Apstein CS, Gravino FN, Haudenschild CC. Determinants of a protective effect of glucose and insulin on the ischemic myocardium. Effects on contractile function, diastolic compliance, metabolism, and ultrastructure during ischemia and reperfusion. Circ Res 1983;52:515–26. 5. Fath-Ordoubadi F, Beatt KJ. Glucose-insulin-potassium therapy for treatment of acute myocardial infarction. An overview of randomized placebo-controlled trials. Circulation 1997;96:1152– 6. 6. Taegtmeyer H, Roberts AFC, Rayne AEG. Energy metabolism in reperfused rat heart: return of function before normalization of ATP content. J Am Coll Cardiol 1985;6: 864–70. 7. Kudo N, Barr AJ, Barr RL, Desai S, Lopaschuk GD. High rates of fatty acid oxidation during reperfusion of ischemic hearts are associated with a decrease in malonyl-CoA levels due to an increase in 59-AMP-activated protein kinase inhibition of acetyl-Coa carboxylase. J Biol Chem 1995;270: 17513–20. 8. Goodwin GW, Taegtmeyer H. Metabolic recovery of the isolated working rat heart after brief global ischemia. Am J Physiol 1994;267:H462–70. 9. Taegtmeyer H, Hems R, Krebs HA. Utilization of energy providing substrates in the isolated working rat heart. Biochem J 1980;186:701–11. 10. Goodwin GW, Arteaga JA, Taegtmeyer H. Glycogen turnover in the isolated working rat heart. J Biol Chem 1995;270: 9234– 40. 11. Doenst T, Guthrie PH, Chemnitius J-M, Zech R, Taegtmeyer H. Fasting, lactate, and insulin improve ischemia tolerance: a comparison with ischemic preconditioning. Am J Physiol 1996;270:H1607–15. 12. Zar J. Biostatistical analysis, 2nd ed. Inglewood Cliffs: Prentice-Hall, 1984.
Ann Thorac Surg 1999;67:1682– 8
13. Schneider CA, Taegtmeyer H. Fasting in vivo delays myocardial cell damage after brief periods of ischemia in the isolated working rat heart. Circ Res 1991;68:1045–50. 14. Depre C, Hue L. Inhibition of glyocogenolysis by a glucose analogue in the working rat heart. J Mol Cell Cardiol 1997;29: 2253–9. 15. Ferranini E, Santoro D, Bonadonnar R, Natali A, Parodi O, Camici P. Metabolic and hemodynamic effects of insulin on human hearts. Am J Physiol 1993;264:E308 –15. 16. Baron AD. Hemodynamic actions of insulin. Am J Physiol 1994;267:E187–222. 17. Barrett E, Schwartz R, Francis C, Zaret B. Regulation by insulin of myocardial glucose and fatty acid metabolism in the conscious dog. J Clin Invest 1984;74:1073–9. 18. Scherrer U, Sartori C. Insulin as a vascular and sympathoexcitatory hormone. Implications for blood pressure regulation, insulin sensitivity, and cardiovascular morbidity. Circulation 1997;96:4104–13. 19. Taegtmeyer H, Goodwin GW, Doenst T, Frazier OH. Substrate metabolism as a determinant for postischemic functional recovery of the heart. Am J Cardiol 1997;80:3A–10A. 20. Hendrickson SC, St. Louis JD, Lowe JE, Abdel-aleem S. Free fatty acid metabolism during myocardial ischemia and reperfusion. Mol Cell Biochem 1997;166:85–94. 21. Gamble J, Lopaschuk GD. Insulin inhibition of 59 adenosine monophosphate–activated protein kinase in the heart results in activation of acetyl coenzyme A carboxylase and inhibition of fatty acid metabolism. Metabolism 1997;46:1270– 4. 22. Mallet RT, Hartman DA, Bu¨nger R. Glucose requirement for postischemic recovery of perfused working heart. Eur J Biochem 1990;188:481–93. 23. Bolukoglu H, Goodwin GW, Guthrie PH, Carmical SG, Chen TM, Taegtmeyer H. Metabolic fate of glucose in reversible low-flow ischemia of the isolated working rat heart. Am J Physiol 1996;270:H817–26. 24. Lagerstrom CF, Walker WE, Taegtmeyer H. Failure of glycogen depletion to improve left ventricular function of the rabbit heart after hypothermic ischemic arrest. Circ Res 1988;63:81– 6. 25. McElroy DD, Walker WE, Taegtmeyer H. Glycogen loading improves left ventricular function of the rabbit heart after hypothermic ischemic arrest. J Appl Cardiol 1989;4:455– 65. 26. Chin E, Allen D. Effects of reduced muscle glycogen concentration on force, Ca21 release and contractile protein function in intact mouse skeletal muscle. J Physiol (Lond) 1997; 498:17–29.