Contribution of Glycogen and Exogenous Glucose to Glucose Metabolism During Ischemia in the Hypertrophied Rat Heart
Abstract Although hypertrophied hearts have increased rates of glycolysis under aerobic conditions, it is controversial as to whether glucose metabolism during ischemia is altered in the hypertrophied heart. Because endogenous glycogen stores are a key source of glucose during ischemia, we developed a protocol to label the glycogen pool in hearts with either [3H]glucose or [14C]glucose, allowing for direct measurement of both glycogen and exogenous glucose metabolism during ischemia. Cardiac hypertrophy was produced in rats by banding the abdominal aorta for an 8-week period. Isolated hearts from aortic-banded and sham-operated rats were initially perfused under substrate-free conditions to decrease glycogen content to 40% of the initial pool size. Resynthesis and radiolabeling of the glycogen pool with [3H]glucose or [14C]glucose were accomplished in working hearts by perfusion for a 60-minute period with 11 mmol/L [3H]glucose or [14C]glucose, 0.5 mmol/L lactate, 1.2 mmol/L palmitate, and 100 μmol/mL insulin. Although glycolytic rates during the aerobic perfusion were significantly greater in hypertrophied hearts compared with control hearts, glycolytic rates from exogenous glucose were not different during low-flow ischemia. The contribution of glucose from glycogen was also not different in hypertrophied hearts compared with control hearts during ischemia (1314±665 versus 776±310 nmol · min−1 · g dry wt−1, respectively). Glucose oxidation rates decreased during ischemia but were not different between the two groups. However, in both hypertrophied and control hearts, the ratio of glucose oxidation to glycolysis was greater for glucose originating from glycogen than from exogenous glucose. Our data demonstrate that glycogen is a significant source of glucose during low-flow ischemia, but the data do not differ between hypertrophied and control hearts.
Pressure overload–induced myocardial hypertrophy is associated with alterations in both fatty acid and glucose metabolism.1 2 3 4 A key metabolic alteration that exists in aerobically perfused hypertrophied hearts is an increased rate of glycolysis,4 5 which may persist into ischemia.6 7 Despite the fact that glycolysis has been directly shown to be accelerated under aerobic conditions, it has also been suggested that the hypertrophied heart may have a decreased potential for glycolysis under hypoxic conditions8 and a reduced capacity to recruit anaerobic glycolysis during ischemia and reperfusion.9 In a recent study, we demonstrated that the hypertrophied heart does not have a depressed capacity to recruit glycolysis or oxidative metabolism after global no-flow ischemia.5 10 However, direct measurements of overall glucose utilization during ischemia in the hypertrophied heart have not been made.
Glucose catabolism in the heart consists of two distinct pathways, glycolysis and mitochondrial glucose oxidation. Although the majority of ATP produced from glucose catabolism in the heart is normally derived from glucose oxidation, during ischemia glucose oxidation decreases and glycolysis becomes a more important source of ATP production. The extent by which glucose oxidation decreases depends primarily on the severity of the ischemic insult. In order to measure both glycolysis and glucose oxidation in the ischemic heart, we have previously perfused isolated working rat hearts with [3H]glucose and [14C]glucose, which allows for the direct measure of glycolysis and glucose oxidation, respectively.10 However, during ischemia, glycogen is mobilized and becomes a major source of glucose for glycolysis. The breakdown of glycogen during ischemia is initiated by a series of steps that leads to the phosphorylation and activation of phosphorylase a.11 12 13 Therefore, measurements of overall glucose metabolism during ischemia need to consider both exogenous and endogenous sources of glucose.
Since glycolytic rates are increased in the hypertrophied heart during aerobic perfusion4 5 10 and since glycolytic isoenzyme content is increased,1 14 it is reasonable to suggest that during ischemia glycolytic rates will also be accelerated. Although indirect measurements suggest that hypertrophied hearts have accelerated rates of glycolysis during ischemia,15 it has not been determined directly whether the differences in glycolytic rates seen under aerobic conditions extend into ischemia. Previous indirect measurements suggest that glycolysis is inhibited to a greater extent during low-flow ischemia.9 However, direct measurements of glycolysis have not been made, and there may exist a situation in which key enzymes for glycolysis (such as phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase) are differently regulated during ischemia in hypertrophied and control hearts. Furthermore, even though oxygen availability is markedly decreased during ischemia, some oxidative metabolism still persists at very low flow rates. Although both glucose oxidation and fatty acid oxidation decrease during ischemia, it is not clear whether these rates decrease to the same extent in control and hypertrophied hearts. Despite the fact that studies have implicated both a decrease in glycolytic potential9 and an increase in glycolysis5 10 as important contributing factors to the sequelae of ischemic injury in the hypertrophied heart, direct measurements of overall glucose metabolism remain to be determined.
To determine the contribution of glycogen and exogenous glucose to both glycolysis and residual glucose oxidation during ischemia, we developed a protocol that allowed us to radiolabel the glycogen pools of normal and hypertrophied rat hearts in vitro. Using a differential radiolabeling technique, we were able to directly measure the contribution of exogenous glucose and glycogen to both glycolysis and glucose oxidation rates during low-flow ischemia. These studies were performed in isolated working hearts obtained from control and aortic-banded rats.
Materials and Methods
d-[5-3H]Glucose and d-[U-14C]glucose were purchased from Du Pont-New England Nuclear. Bovine serum albumin (fraction V) was obtained from Boehringer-Mannheim. Hyamine hydroxide (methylbenzethonium, 1 mol/L in methanol solution) was obtained from Sigma Chemical Co. Dowex 1-X4 anion exchange resin (200- to 400-mesh chloride form) was obtained from Bio-Rad. ACS counting scintillant was purchased from Amersham Canada Ltd. Ecolite counting scintillant was obtained from ICN Biomedicals Canada Ltd. All other chemicals were reagent grade.
Pressure-overload cardiac hypertrophy was produced as previously described.4 Briefly, male Sprague-Dawley rats weighing 60 to 70 g (3 weeks of age) were anesthetized with a 50 mg/kg intraperitoneal injection of methohexital sodium. A lateral incision of the abdominal wall was used to expose the abdominal aorta. The aorta was isolated, and a minimally occlusive (0.4-mm) hemoclip was applied in a suprarenal position. In control animals, the aorta was isolated but not banded. The incision was then closed, and the animals were allowed to recover for an 8-week period. After the 8-week period, the animals were anesthetized with sodium pentobarbital (60 mg/kg IP), and the hearts were quickly removed for study.
Glycogen Depletion and Resynthesis/Labeling
Both control and hypertrophied hearts were exposed to the same perfusion protocols (Fig 1⇓). After the animals were anesthetized, the hearts were removed and immediately cannulated, and an aortic retrograde perfusion was initiated. The left atrium was subsequently cannulated to allow for future use in the working mode. The Langendorff perfusate consisted of a Krebs-Henseleit buffer (pH 7.4) gassed with a 95% O2/5% CO2 air mixture, with a free Ca2+ concentration of 2.50 mmol/L and 11 mmol/L glucose. To deplete the hearts of glycogen, a 30-minute period of aortic retrograde perfusion was subsequently initiated with a buffer similar to the one mentioned above, but no exogenous glucose or any other carbon substrate was present. Perfusion pressure was maintained at a constant 60 mm Hg throughout the depletion protocol. A series of hearts were also frozen at the end of the 30-minute depletion period to measure tissue glycogen, lactate, and high-energy phosphates (not shown in Fig 1⇓).
After the depletion protocol, the hearts were subsequently switched to a working mode, which allowed for left ventricular filling via the left atrium. The perfusate was also a Krebs-Henseleit solution (pH 7.4) gassed with a 95% O2/5% CO2 air mixture, with a free Ca2+ concentration of 2.50 mmol/L and 11 mmol/L glucose, but now the buffer also included 0.5 mmol/L lactate, 1.2 mmol/L palmitate prebound to 3% bovine serum albumin, and 100 μU/mL insulin. Perfusion pressures of 11.5 mm Hg left atrial preload and 80 mm Hg aortic afterload were used. Hearts were perfused for 60 minutes to resynthesize the glycogen pool.
As shown in Fig 1⇑, the protocol to label the glycogen pool in the control and hypertrophied hearts involved the addition of [3H]glucose (series A) or [14C]glucose (series C) to the working hearts after the initial 30-minute Langendorff perfusion period. In series A, the glycogen pool was labeled with [3H]glucose, allowing for measurements of total glycolytic flux during the low-flow ischemic period. The addition of [14C]glucose to the perfusate at the time of low-flow ischemia allowed for the measurement of glucose oxidation from exogenous glucose only, since the glycogen pool was not prelabeled with [14C]glucose. The addition of [14C]glucose after the initial Langendorff period (series C) resulted in a labeling of the glycogen pool with [14C]glucose, allowing for the measurements of total rates of glucose oxidation during the low-flow ischemic period (series B). In these hearts, addition of [3H]glucose to the perfusate at the onset of low-flow ischemia allowed us to measure glycolytic flux from exogenous glucose only.
Perfusate buffer samples required for metabolic analysis were taken at 10, 20, 40, and 60 minutes during the resynthesis period.
In a separate series of perfusions, a group of hearts were subjected to glycogen depletion, and either glycolysis or glucose oxidation was measured individually during the resynthesis period. Hearts from anesthetized (halothane, 2% to 3%) control and aortic-banded rats were initially perfused via the aorta in the Langendorff mode with substrate-free insulin-free Krebs-Henseleit solution for 30 minutes to deplete myocardial glycogen. Hearts were then switched to the working mode and perfused with recirculating Krebs-Henseleit solution containing either (1) 11 mmol/L [5-3H]glucose, 0.5 mmol/L lactate, 1.2 mmol/L palmitate prebound to 3% albumin, and 100 μU/mL insulin or (2) 11 mmol/L [U-14C]glucose, 0.5 mmol/L lactate, 1.2 mmol/L palmitate prebound to 3% albumin, and 100 μU/mL insulin. Glycolysis and glucose oxidation were measured over the 60-minute resynthesis period.
Low-Flow Ischemia Protocol
Upon switching the hearts to low-flow ischemia (see Fig 1⇑), which was initiated by an aortic retrograde perfusion of 0.5 mL/min, the other radiolabel of glucose was added to the perfusion system so that dual labeling now occurred; ie, series A consisted of [3H]glucose during resynthesis followed by [14C]glucose added at the start of low flow, and series C consisted of [14C]glucose during resynthesis followed by [3H]glucose added at the start of low flow. Series A hearts ([3H]glucose followed by [14C]glucose) allowed us to additionally measure the contribution of exogenous glucose to glucose oxidation during low-flow ischemia, whereas series C hearts ([14C]glucose followed by [3H]glucose) allowed for the additional measurement of glycolysis from exogenous glucose during low-flow ischemia.
To calculate the contribution of glycogen to glycolysis during low-flow ischemia, rates of glycolysis from exogenous glucose (series C) were subtracted from rates of total glycolysis taken during low-flow ischemia (series A). Calculation of glucose oxidation from endogenous glycogen during low-flow ischemia was achieved in a similar manner; namely, rates of glucose oxidation from exogenous glucose (series A) were subtracted from rates of total glucose oxidation during low-flow ischemia (series C).
During low-flow ischemia, samples of perfusion buffer were taken at 5, 10, 20, 40, and 60 minutes. Hearts were also frozen at the end of the glycogen-depletion period, at the end of the resynthesis period (series B), and at the end of low-flow ischemia (series A and C) for the measurement of glycogen, lactate, and high-energy phosphate content. Peak systolic pressure during the resynthesis period was recorded on a Grass 79-D physiograph with a Spectramed p 23XL pressure transducer. Cardiac output and coronary flow were measured using Transonics in-line flow probes.
Measurement of Glycolysis and Glucose Oxidation
Total and exogenous glycolysis were measured by determining the amount of 3H2O produced (which is liberated from [5-3H]glucose at the enolase step of glycolysis). Separation of 3H2O from [3H]/[14C]glucose was achieved as described earlier.4 16 Measurement of total and exogenous glucose oxidation was achieved by quantitatively collecting 14CO2 produced (which is liberated from [U-14C]glucose at the level of the pyruvate dehydrogenase complex and in the trichloroacetic acid cycle) as described previously.16 Briefly the 14CO2 produced as a gas was collected from the sealed perfusion system through a 1 mol/L methylbenzethonium hydroxide solution, which acted as a 14CO2 trap. Perfusate samples containing [14C]bicarbonate were collected and stored under mineral oil to prevent the liberation of 14CO2 and subsequently injected into closed metabolic reaction flasks containing 9N H2SO4. The 14CO2 released from the perfusion buffer was trapped in center wells containing filter paper saturated with 1 mol/L methylbenzethonium hydroxide. Afterward, the center wells were removed and counted in ACS scintillant using standard counting procedures. Therefore, total 14CO2 production was determined by analyzing samples of the methylbenzethonium hydroxide used as a 14CO2 gas trap and perfusate samples that contained [14C]bicarbonate.
At the end of the perfusions, heart ventricles were freeze-clamped using Wollenberger clamps cooled to the temperature of liquid nitrogen. The frozen tissue was weighed to determine total wet ventricular weight. The atria were dried, weighed, and used in the calculation of whole-heart weight.
Frozen ventricular tissue was powdered using a mortar and pestle cooled to the temperature of liquid nitrogen. A portion of the tissue was dried and used to calculate the dry-to-wet ratio of the tissue. The remainder of the tissue was stored in a −70°C freezer. The dry weight–to–wet weight ratio as well as the wet ventricular weight and the atrial dry weight were used to calculate the total dry weight of the heart.
ATP and creatine phosphate was measured in heart tissue obtained at the end of ischemia and after reperfusion. The tissue was extracted with a 6% perchloric acid/0.5 mmol/L EGTA solution, followed by centrifugation and neutralization with K2CO3 (5 mol/L). The supernatant was taken and analyzed by HPLC using a modified method of Ally and Park.17
Myocardial lactate was extracted from frozen tissue with 6% perchloric acid. Tissue lactate was then measured using a spectrophotometric assay.18 Glycogen content was measured by alkaline extraction of tissue, ethanol precipitation, and acidic hydrolysis of glycogen to yield free glucose. A spectrophotometric assay was then used to determine the glucose content.18
The data are represented as the mean±SEM. When comparing two group means, a Student’s unpaired two-tailed t test was used. A value of P<.05 was regarded as significant.
Animal Body and Heart Weights
Body and wet heart weights of control and aortic-banded animals (hypertrophied hearts) were measured 8 weeks after the banding procedure was performed. There was a significant increase (21%, P<.05) in heart weight in hypertrophied hearts (2.16±0.23 g, n=38) compared with control hearts (1.78±0.17 g, n=36). This was not accompanied by an increase in body weight after 8 weeks (433±37 versus 433±34 g in aortic-banded and control rats, respectively) and resulted in a significant increase (21%, P<.05) in the heart weight–to–body weight ratio in the aortic-banded animals (5.01±0.50 versus 4.13±0.35 in aortic-banded and control rats, respectively).
Myocardial Function of Control and Hypertrophied Rat Hearts
Functional measurements were taken throughout the glycogen resynthesis period. Recovery of function after the 30-minute depletion period was immediate (Table 1⇓). Control and hypertrophied hearts had similar mechanical parameters after 10 minutes of working heart perfusion, as seen in Table 1⇓. Mechanical function at 10 minutes of resynthesis (measured as heart rate×peak systolic pressure) was similar between control and hypertrophied hearts (28.05±1.01 versus 26.12±0.84 bpm · mm Hg · 10−3, respectively; P=NS). However, measurements of cardiac work (measured as cardiac output×peak systolic pressure) suggested that a depression in the mechanical function of the hypertrophied hearts compared with control hearts existed (55.12±4.24 versus 67.16±4.18 mL · min−1 · mm Hg · 10−2, respectively; P<.05). This decrease in cardiac work was primarily due to the significantly decreased cardiac output of the hypertrophied hearts compared with control hearts. Mechanical function was stable throughout the 60-minute period of glycogen resynthesis for both control and hypertrophied hearts. However, at the end of the 60-minute period, cardiac output, aortic output, and coronary flow were all significantly depressed in the hypertrophied hearts compared with control hearts. Compared with control hearts, the hypertrophied hearts exhibited the same depression in cardiac work at the end of 60 minutes (52.83±3.63 versus 67.92±3.96 mL · min−1 · mm Hg · 10−2, respectively; P<.05).
Cumulative Rates of Glycolysis and Glucose Oxidation From Exogenous Glucose and Endogenous Glycogen
During low-flow ischemia, it is not known if rates of total and exogenous glycolysis exist in a steady state or if they change over the time of the ischemic insult. Plotting cumulative glycolysis from exogenous glucose during the aerobic resynthesis period, total glycolysis during low flow (Fig 2A⇓), and glycolysis from exogenous glucose (Fig 3A⇓) versus the time of aerobic perfusion and low-flow ischemia suggests that rates of glycolysis do not vary over the 60-minute period of glycogen resynthesis or low-flow ischemia.
Rates of exogenous glucose oxidation during the aerobic resynthesis period did not show the same linearity as did the rates of exogenous glycolysis during the same period (Fig 2B⇑). The oxidation of glucose significantly increases in control hearts from 0.518±0.068 to 0.786±0.091 μmol · min−1 · g dry wt−1 (P<.05) between 10 to 20 minutes and between 40 to 60 minutes of aerobic perfusion, respectively. A significant increase of greater magnitude occurs in the hypertrophied hearts from 0.677±0.091 to 1.145±0.124 μmol · min−1 · g dry wt−1 (P<.05) during the same time periods. Because the rates of glucose oxidation do not follow a steady state rate over the 60-minute period of perfusion, the initial rate (10 to 20 minutes of resynthesis) is taken to be indicative of the contribution of exogenous glucose to glucose oxidation. The increase in total glucose oxida-tion at the end of the 60-minute resynthesis period may be due to an increased contribution of glycogen cycling during resynthesis to glucose oxidation.
Cumulative rates of total and exogenous glucose oxidation (Fig 3B⇑) measured during low-flow ischemia exhibited the same linearity as did total and exogenous glycolysis rates during low-flow ischemia. The presence of measurable rates of glucose oxidation during low-flow ischemia suggests that the degree of ischemia, although severe enough to depress function, did not entirely suppress oxidative metabolism. Rates of total glucose oxidation from both endogenous glycogen and exogenous glucose were severely depressed but exhibited a linearity that was similar to what was seen for glycolysis during low-flow ischemia. This suggests that rates of glucose oxidation during low-flow ischemia can be sustained over the 60-minute period.
Steady State Rates of Glycolysis and Glucose Oxidation From Exogenous Glucose and Endogenous Glycogen
Steady state rates of exogenous and endogenous glycolysis and glucose oxidation are shown in Fig 4⇓. These values were calculated from the data in Figs 2⇑ and 3⇑. The linear portion of the curve was used to calculate steady state rates (ie, between 10 and 60 minutes for aerobic calculations and between 5 and 60 minutes during low-flow ischemia). This was done for both the exogenous and endogenous calculations. The first 5 minutes of low-flow ischemia cannot be used, since this is the time necessary for equilibration of the radiolabeled glucose added at the onset of ischemia.
The hypertrophied heart exhibits steady state rates of exogenous glycolysis (Fig 4A⇑) that are greater than rates of glycolysis in control hearts during the aerobic glycogen resynthesis period (4.446±0.305 versus 2.903±0.307 μmol · min−1 · g dry wt−1, respectively; P<.05). These rates of exogenous glycolysis compare favorably to what we have previously seen in other studies of hypertrophied hearts (4.325±0.716 versus 2.313±0.272 μmol · min−1 · g dry wt−1 for hypertrophied and control hearts perfused under similar buffer conditions, respectively; P<.05).10 During low-flow ischemia, we did not see this continued acceleration of glycolysis from exogenous glucose in the hypertrophied heart. Rates of glycolysis from exogenous glucose during low-flow ischemia decreased significantly in the hypertrophied heart to levels that were similar to those in the control hearts (2.593±0.503 versus 2.797±0.276 μmol · min−1 · g dry wt−1, respectively; P=NS). When rates of total glycolysis from glycogen metabolism during low-flow ischemia were calculated (by subtracting rates of glycolysis from exogenous glucose from total rates of glycolysis), we found that glycogen was making a significant contribution to total rates of glycolysis. As Fig 4A⇑ shows, there was a slight acceleration in the rates of glycolysis from glycogen in the hypertrophied heart compared with the control heart; however, it was not of statistical significance (1.314±0.665 versus 0.779±0.310 μmol · min−1 · g dry wt−1, respectively; P=NS).
It should be noted that substrate cycling in the glycogen pool was not taken into account during low-flow ischemia, since the fate of unlabeled glucose could not be followed. However, the fact that the cumulative curves during low-flow ischemia are linear suggests that there was not a dilution of the labeled glycogen or exogenous glucose pool, because any alteration by the unlabeled pool would have resulted in a nonlinear cumulative curve.
When rates of glycogen depletion were calculated over the 60-minute period of low-flow ischemia by subtracting the glycogen content at the end of ischemia from the glycogen content after resynthesis (from Table 2⇓), these calculated rates of glycogen breakdown were similar compared with those measured using our technique of glycogen labeling. In control hearts, the calculated rates of glycogen use were 1.16±0.19 versus 0.78±0.31 μmol · min−1 · g dry wt−1 for the direct measurement. In hypertrophied hearts, the values were also similar, with the calculated rate of glycogen use being 1.03±0.15 versus 1.31±0.67 μmol · min−1 · g dry wt−1 for the direct measurement used in the present study (P>NS).
During the early period of aerobic resynthesis (10 to 20 minutes) (Fig 4B⇑), steady state rates of glucose oxidation were similar in the hypertrophied hearts compared with control hearts (0.677±0.091 versus 0.518±0.068 μmol · min−1 · g dry wt−1, respectively; P=NS). These rates of exogenous glucose oxidation are significantly higher than what we have previously measured in both control and hypertrophied hearts (0.198±0.020 and 0.135±0.044 μmol · min−1 · g dry wt−1, respectively; P<.05).10 During low-flow ischemia, steady state rates of glucose oxidation decrease considerably; however, there remain measurable rates of glucose oxidation from both glycogen and exogenous glucose. In control hearts, rates of glucose oxidation from exogenous glucose compared with endogenous glycogen were 0.175±0.031 and 0.155±0.059 nmol · min−1 · g dry wt−1, respectively (P=NS). Similar rates of glucose oxidation from exogenous glucose and endogenous glycogen were seen in the hypertrophied heart (0.168±0.053 and 0.197±0.094 nmol · min−1 · g dry wt−1, respectively; P=NS).
Metabolite Content of Control and Hypertrophied Hearts After Glycogen Depletion, Resynthesis, and Low-Flow Ischemia
Table 2⇑ shows the glycogen and lactate content of the control and hypertrophied hearts after the 30 minutes of glycogen depletion, 60 minutes of glycogen resynthesis and labeling, and 60 minutes of low-flow ischemia. Glycogen levels in freshly frozen, unperfused hearts were found to be 116.1±11.9 and 126.6±12.2 μmol/g dry wt−1 for control and hypertrophied hearts, respectively (P=NS). Glycogen was successfully resynthesized from a depleted state after 60 minutes of perfusion in the presence of 1.2 mmol/L palmitate, 0.5 mmol/L lactate, and 11 mmol/L glucose, as seen in Table 2⇑. The percentage of the glycogen pool that was labeled was similar in both the control and hypertrophied hearts at the end of the resynthesis period (61.6±4.3% and 54.9±6.4%, respectively). At the end of the low-flow period, the glycogen pool dropped to similar levels in both control and hypertrophied hearts. The glycogen pool at the end of low-flow ischemia was labeled to an extent similar to that seen after the resynthesis period (52.3±4.4% and 62.0±7.6% of the glycogen pool in control and hypertrophied hearts, respectively). There was also incorporation of the label added during the low-flow period into the glycogen pool, suggesting glycogen synthesis during low-flow ischemia (16.1±1.8% and 24.1±3.3% of the glycogen pool in control and hypertrophied hearts, respectively). Although a similar percentage of label was seen at the end of low-flow ischemia, it needs to be recognized that radiolabel was also being replaced back into the glycogen pool as the glycogen was being catabolized. Our results do not correct for rates of reincorporation of glucose in calculating rates of glycogen metabolism. However, no differences in glycogen pool size, glycogen labeling, or glycogen relabeling before or at the end of ischemia were observed in control and hypertrophied hearts. Rates of glycogen catabolism were also similar. Therefore, it is unlikely that differences occurred in the amount of unlabeled glucose metabolism between the control and hypertrophied hearts.
To determine if differences in coronary flow between control and hypertrophied hearts could explain both the differences in glycolytic rates during the resynthesis period and differences in lactate levels at the end of the resynthesis period, a group of hearts were subjected to the glycogen-depletion protocol period, and glycolysis and glucose oxidation rates were measured individually. In these hearts, lactate levels at the end of the resynthesis period were not different (7.7±0.8 and 6.7±1.1 μmol/g dry wt in control [n=12] and hypertrophied hearts [n=12], respectively; P=NS). In these hearts, glycolytic rates were also elevated in hypertrophied hearts during resynthesis (rates were 2695±55 and 3745±64 nmol · min−1 · g dry wt−1 in control hearts [n=6] and hypertrophied hearts [n=6], respectively; P<.05). Glucose oxidation rates were not different between the two groups during the resynthesis period (466±26 and 497±20 nmol · min−1 · g dry wt−1, respectively; P=NS). This suggests that the high glycolytic rates during resynthesis were a fundamental difference between control and hypertrophied hearts and were not due to the small differences in coronary flow.
Lactate content at the end of the 30-minute glycogen depletion was similar in both control and hypertrophied hearts (Table 2⇑). At the end of the 60-minute resynthesis period, there was a greater tissue lactate content in the hypertrophied hearts than in the control hearts. This may have occurred because of the lower coronary flow rates in hypertrophied hearts compared with control hearts (22±2 versus 28±1 mL/min, P<.05) coupled with accelerated glycolysis. There was also a significant accumulation of lactate in the hypertrophied hearts compared with control hearts at the end of the 60-minute period of low-flow ischemia.
ATP values were not different in either the control or hypertrophied hearts at the end of 30-minute depletion period (11.1±1.3 versus 9.5±0.9 μmol/g dry wt, respectively; P=NS), nor were levels of creatine phosphate (16.8±2.3 versus 14.2±1.3 μmol/g dry wt for control and hypertrophied hearts, respectively; P<.05). The 60-minute period of glycogen resynthesis did not increase ATP or creatine phosphate content in control hearts (10.4±1.4 and 18.6±3.7 μmol/g dry wt, respectively; P=NS). However, hypertrophied hearts saw an increase in creatine phosphate (from 14.2±1.3 to 20.0±1.8 μmol/g dry wt, P<.05). ATP content did not change in the hypertrophied hearts (10.3±0.9 μmol/g dry wt) at the end of the 60-minute resynthesis period. At the end of low-flow ischemia, both ATP and creatine phosphate levels dropped in control hearts (8.0±1.4 and 13.4±2.6 μmol/g dry wt, respectively) and in hypertrophied hearts (7.4±1.2 and 10.0±2.7 μmol/g dry wt, respectively).
We have previously shown that the aerobically perfused hypertrophied hearts have increased rates of glycolysis compared with normal hearts.4 This increase in glycolysis has been shown again in the present study, even when glycogen synthesis was occurring. The rates of glycolysis obtained is the present study were also comparable to what has previously been shown to exist in other studies.4 5 10 The observed increase in glycolysis from exogenous glucose may be linked to the upregulation of some key glycolytic enzymes,1 14 or it may be an attempt to normalize ATP production due to decreased ATP supply from fatty acid β-oxidation.4 A clear understanding of whether rates of glycolysis are altered during ischemia in the hypertrophied heart is important, since it has been suggested that the hypertrophied heart has a decreased ability to recruit anaerobic glycolysis and that this may be detrimental during postischemic reperfusion8 9 ; however, other studies have suggested that the hypertrophied heart actually has accelerated rates of glycolysis during ischemia.6 15 The source of glucose during ischemia may be exogenous glucose if a residual coronary flow exists, but endogenous glycogen can also significantly contribute glucose for glycolysis. Before the present study, the exact contribution of glycogen to energy metabolism during low-flow ischemia was not definitively known.
The present data define the contribution of both endogenous glycogen and exogenous glucose during a low-flow (0.5 mL/min) ischemia to rates of glycolysis and glucose oxidation. Rates of glycolysis during low-flow ischemia were no longer increased in hypertrophied hearts as they were during aerobic perfusion.4 5 10 We have also found that there was a significant glycogen component to total rates of glycolysis that could readily be measured by our model of glycogen depletion and resynthesis in both control and hypertrophied hearts. The contribution of glycogen to glycolysis during low-flow ischemia may be slightly accelerated in hypertrophied hearts compared with normal hearts, constituting 33% and 24%, respectively, of the total glycolytic rates during low-flow ischemia. However, during a low-flow ischemia, the hypertrophied heart does not have increased rates of glycolysis beyond that of a normal heart. Why glycolytic rates were accelerated during the aerobic perfusion and not during low-flow ischemia has yet to be completely delineated. One possibility is that despite having an increased expression and/or activity of glycolytic enzymes in the hypertrophied hearts, these enzymes are subjected to the same degree of feedback inhibition during ischemia. It is also possible that the higher degree of lactate accumulation during ischemia in the hypertrophied hearts may inhibit glycolysis to a greater extent in the hypertrophied hearts than in the control hearts and may account for the fact that rates were no longer accelerated during ischemia.
Rates of glucose oxidation were also measured in the control and hypertrophied hearts during aerobic perfusion and low-flow ischemia. We found that rates of glucose oxidation during the preischemic labeling period were significantly higher than what we had previously seen in control and hypertrophied hearts perfused under similar buffer conditions.5 10 This may have been due to the decreased contribution of glycogen to glucose oxidation. The model of low-flow ischemia that we used effectively abolished mechanical function and resulted in a moderately reduced oxidative metabolism. Rates of glucose oxidation from exogenous glucose dropped significantly compared with the preischemic values. However, glycogen metabolism played a significant role in supplying residual oxidative metabolism with glucose for glucose oxidation.
It is possible that differences in coronary flow between control and hypertrophied hearts could explain the difference in glycolytic rates during the resynthesis period and the lactate levels at the end of the resynthesis period or that hypertrophied hearts may be responding differently to the reintroduction of glucose following glycogen depletion. The second scenario is unlikely, since we have previously reported high glycolytic rates in hypertrophied hearts in which the glycogen pool has not been previously depleted.4 10 The first scenario is also unlikely, since we subjected a second group of hearts to the glycogen depletion and resynthesis protocol and found no differences in lactate levels at the end of the resynthesis period. Despite the lack of difference in lactate levels, these hearts also had elevated rates of glycolysis during the resynthesis period. Furthermore, the difference in lactate levels between control and hypertrophied hearts at the end of the resynthesis period shown in Table 2⇑ was quite small (4.3±0.6 and 10.8±1.8 μmol/g dry wt, respectively). It should be recognized that total glycolysis during the 60-minute resynthesis period was 280 μmol/g dry wt in hypertrophied hearts and 180 μmol/g dry wt in control hearts (see Fig 2⇑). These values were comparable to total glucose oxidation rates over the same period (60 μmol/g dry wt in hypertrophied hearts and 40 μmol/g dry wt in control hearts) (see Fig 2⇑). As a result, glycolysis uncoupled from glucose oxidation over the resynthesis period was 220 μmol/g dry wt in hypertrophied hearts and 140 μmol/g dry wt in control hearts. This would mean that lactate production was ≈160 μmol/g dry wt greater in hypertrophied hearts than in control hearts during the resynthesis period: (220−140)×2 lactate from each molecule of glucose passing through glycolysis that is not oxidized. As a result, the slightly higher lactate levels at the end of the resynthesis period could simply be due to differences in lactate production as a result of the increased glycolysis rates in the hypertrophied hearts.
The data indicate that nearly 20% of the glucose from glycogen passing through glycolysis in control hearts and 15% of that in hypertrophied hearts goes to residual oxidative metabolism. These values are comparable to 6% of exogenous glucose passing through glycolysis going to residual glucose oxidation for both control and hypertrophied hearts during low-flow ischemia. This preferential usage of glucose from glycogen is not unlike what has previously been shown in carotid artery smooth muscle. Hardin and Kushmerick19 suggest that in smooth muscle, glycogen-derived pyruvate passes preferentially into glucose oxidation. We have recently shown that during aerobic perfusion of isolated working rat hearts, a preferential use of glucose from glycogen for glucose oxidation exists.20 This finding strengthens the implications made by Hardin and Kushmerick, who suggested that pyruvate formed from exogenous glucose via glycolysis and pyruvate from glycogen metabolism do not form a homogeneous intracellular pyruvate pool. The present data support this concept in the heart during aerobic perfusion20 and even during a period of low-flow ischemia, in which oxidative metabolism is moderately depressed. In addition to channeling of pyruvate from endogenous and exogenous glucose, it should be recognized that channeling of exogenous pyruvate may also occur. Studies by Lewandowski21 and Bunger22 have shown that perfusion of hearts with high concentrations of pyruvate will result in preferential oxidation of exogenous pyruvate over that of pyruvate from endogenous sources. This raises the possibility of a third source of pyruvate channeling. To date, it has yet to be determined what effect exogenously added pyruvate has on pyruvate derived from exogenous or endogenous glucose.
As well, it is interesting to note that rates of glucose oxidation increase during the 60-minute resynthesis period in control and hypertrophied hearts. This may be due to a greater cycling of glucose from glycogen into glucose oxidation as the glycogen pool increases in size (and is 14C-labeled) during resynthesis. Further evidence for this is based on the greatly enhanced rates of glucose oxidation that we see in both control and hypertrophied hearts during the aerobic resynthesis period. Compared with other studies in which hearts were perfused under similar conditions,5 10 we have found that rates of glucose oxidation are 4- to 5-fold greater in the present study. This suggests that greater amounts of pyruvate from exogenous glucose passing through glycolysis are proceeding to glucose oxidation. Why this occurs may be due to the depleted glycogen pool, which, because it is being resynthesized, may not contribute to glucose oxidation to such a great extent. As the glycogen pool increases in size, increased rates of glucose oxidation at the end of the 60-minute aerobic period may come from the increased turnover of 14C-labeled glycogen and its preferential support of glucose oxidation.
Depletion of myocardial glycogen stores was not significantly different in hypertrophied hearts compared with control hearts at the end of the low-flow ischemic period. This supports the data in which we directly measured the glycogen breakdown with the radiolabeled glucose. We found that rates of glycolysis from glycogen and exogenous glucose were linear during low-flow ischemia. The residual labeling of the glycogen pool at the end of low-flow ischemia indicated that the radiolabel was not totally removed from the glycogen pool. The fact that the glycogen pool at the end of low-flow ischemia was labeled to a similar extent as that at the end of the resynthesis period suggests that the labeled glucose units of the glycogen pool were not randomly metabolized. The radiolabeling data also indicate that there may be a minor component of glycogen resynthesis during the low-flow period. We found that at the end of low-flow ischemia, 16.1±1.8% of the glycogen pool in control hearts was labeled with the radioisotope of glucose that was present only during low-flow ischemia. A value of 24.1±3.3% of the glycogen pool in hypertrophied hearts was labeled with the radioisotope of glucose present only during low-flow ischemia. It is clearly evident that the glycogen phosphorylase reaction is greatly exceeding the glycogen synthase reaction, because a net glycogen breakdown does occur, and there is a measurable difference in the rates of total glycolysis and rates of glycolysis from exogenous glucose. However, the implications are that simultaneous glycogen breakdown and synthesis occur in a low-flow ischemia. Also, we may actually be underestimating the rate of glycogen breakdown in the heart.
Compared with rates of glucose oxidation, elevated rates of glycolysis have been linked to depressed postischemic functional recovery of normal hearts.23 24 Uncoupling glucose oxidation from glycolysis leads to H+ production.23 25 26 The hydrolysis of ATP generates one H+ atom, regardless of whether it is derived from glycolysis or mitochondrial metabolism. However, during the generation of glycolytically derived ATP, one H+ atom from glucose is incorporated into ATP, resulting in the production of one H+ atom when the ATP is hydrolyzed. In contrast, the overall flux of glucose through glycolysis and pyruvate oxidation puts as many H+ atoms onto ATP during synthesis as are released during ATP hydrolysis (ie, from pyruvate to ATP, a net negative balance of one H+ atom exists). As a result, the process is H+ neutral.26
An uncoupling of glucose metabolism occurs in the aerobically perfused heart and is due to the inhibition of the pyruvate dehydrogenase complex by a high intramitochondrial acetyl coenzyme A–to–coenzyme A ratio produced by the preferred oxidation of fatty acids.27 Therefore, because of the inhibition of the pyruvate dehydrogenase complex by fatty acid β-oxidation, an imbalance between glucose oxidation and glycolysis leads to a net production of H+ from the hydrolysis of glycolytically derived ATP.23 25 26 The excess production of H+ leads to an impaired postischemic mechanical function in normal hearts23 and may be involved in the depression of postischemic reperfusion recovery in hypertrophied hearts.5 10
It has been suggested an accelerated rate of glycolysis in the hypertrophied heart during ischemia may also result in an increased rate of H+ production due to enhanced rates of glycolysis5 10 as well as glycolytic metabolite accumulation.1 6 15 However, the present study would suggest otherwise, at least during a low-flow ischemia. We have shown that rates of glycolysis and residual oxidative metabolism from exogenous sources and glycogen are similar in hypertrophied hearts compared with normal hearts. This would therefore result in a similar rate of ATP production and net H+ production from the hydrolysis of glycolytically derived ATP.25 Total rates of H+ production from both endogenous glycogen and exogenous glucose yield 7.116±1.097 versus 8.202±2.255 μmol · min−1 · g dry wt−1 for control and hypertrophied hearts, respectively (P=NS).
Recently, we suggested that rates of glycolysis substantially increased above rates of glucose oxidation in the hypertrophied heart, resulting in a significantly increased production of H+ during normal aerobic perfusion and during postischemic reperfusion and, therefore, resulting in H+ overload.5 10 It has been shown that the Na+-H+ exchanger message is increased in the hypertrophied heart,28 suggesting that the hypertrophied heart is upregulating systems that are used to handle the acidosis produced by enhanced glycolytic rates. The Na+-H+ exchanger may be involved in postischemic injury due to subsequent Ca2+ overload from Na+-Ca2+ exchange.29 30 Therefore, increased rates of H+ production in the hypertrophied heart due to accelerated rates of glycolysis during ischemia may result in an exacerbated Na+-H+ and Na+-Ca2+ activity and subsequent Ca2+ overload during early reperfusion. We believe that the contribution of ischemically derived H+ to reperfusion injury is not increased in the hypertrophied heart above normal hearts during low-flow ischemia. However, what occurs during a more severe low-flow or no-flow ischemia may be different, as suggested by a number of other studies.1 6 15 The present study was limited to low-flow ischemia, so that metabolism could be measured by the washout of glycolytic metabolites (3H2O), which is not possible during no-flow ischemia.
In summary, this model of glycogen depletion, resynthesis, and labeling allows us to directly follow the fate of glycogen during low-flow ischemia. We have found that total rates of glycolysis are not enhanced in the hypertrophied heart during low-flow ischemia. Similarly, rates of exogenous glucose use are not accelerated in the hypertrophied heart compared with control hearts, unlike what is seen during normal aerobic perfusion. We have also determined that glycogen contributes a significant percentage to total rates of glycolysis during low-flow ischemia and that simultaneous synthesis and degradation occurs during ischemia, even though catabolism dominates.
This study was supported by the Medical Research Council of Canada. Dr Lopaschuk is a Medical Research Council Scientist and an Alberta Heritage Foundation for Medical Research Senior Scholar. Dr Allard is a Research Scholar of the Heart and Stroke Foundation of Canada. B.O. Schönekess was a graduate student trainee of the Alberta Heritage Foundation for Medical Research and the Heart and Stroke Foundation of Canada. S.L. Henning is a graduate student trainee of the Heart and Stroke Foundation of Canada.
Reprint requests to Dr Gary D. Lopaschuk, 423 Heritage Medical Research Bldg, The University of Alberta, Edmonton, Alberta, Canada T6G 2S2.
- Received October 25, 1996.
- Accepted July 11, 1997.
- © 1997 American Heart Association, Inc.
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