Propionyl l-Carnitine Improvement of Hypertrophied Heart Function Is Accompanied by an Increase in Carbohydrate Oxidation
Abstract Propionyl l-carnitine (PLC) is a naturally occurring derivative of l-carnitine that can improve hemodynamic function of hypertrophied rat hearts. The mechanism(s) responsible for the beneficial effects of PLC is not known, although improvement of myocardial energy metabolism has been suggested. In this study, we determined the effect of PLC on carbohydrate and fatty acid metabolism in hypertrophied rat hearts. Myocardial hypertrophy was produced by partial occlusion of the suprarenal aorta of juvenile rats. Over a subsequent 8-week period, a mild hypertrophy developed, resulting in a 17% increase in heart weight in these animals compared with the sham-operated control animals. Myocardial carnitine was decreased in hypertrophied hearts compared with hearts from sham-operated animals (4155±383 versus 5924±570 nmol · g dry wt−1, respectively; P≤.05). Perfusion of isolated working hearts for 60 minutes with buffer containing 1 mmol/L PLC increased carnitine content in hypertrophied hearts from 4155±383 to 7081±729 nmol · g dry wt−1 (P≤.05). In the presence of 1.2 mmol/L palmitate, fatty acid oxidation rates were not decreased in the hypertrophied hearts compared with control hearts. PLC treatment did not alter rates of fatty acid oxidation in control hearts but did result in a small increase in rates in the hypertrophied hearts. The most dramatic effect of PLC treatment in hypertrophied hearts was an increase in glucose oxidation rates from 137±25 to 627±110 nmol · min−1 · g dry wt−1 (P≤.05) and an increase in lactate oxidation rates from 119±17 to 252±47 nmol · min−1 · g dry wt−1 (P≤.05). Glycolytic rates, which were already significantly elevated in hypertrophied hearts compared with control hearts, were not altered by PLC treatment. Overall ATP production from exogenous sources was increased by 64% in PLC-treated hypertrophic hearts and was accompanied by a significant increase in cardiac work. The main effect of PLC treatment was to increase the contribution of glucose oxidation to the relative rate of ATP production from 11.6% to 21.6%. The contribution of glucose and palmitate oxidation to ATP production was also determined in aortic-banded animals treated with 60 mg/kg PLC for an 8-week period. This treatment was also associated with a significant improvement in mechanical function in hearts isolated from these animals compared with untreated animals as well as an increase in the contribution of glucose oxidation to ATP production. Despite this improvement of cardiac work after chronic PLC treatment, no increase in palmitate oxidation was observed in hypertrophied hearts. These findings indicate that the beneficial effects of PLC in hypertrophied hearts can be accounted for by a stimulation of ATP production from carbohydrate oxidation rather than from fatty acid oxidation. The increase in carbohydrate oxidation may be a consequence of activation of the pyruvate dehydrogenase complex, by means of a reduction in the ratio of intramitochondrial acetyl coenzyme A to coenzyme A.
Pressure-overload hypertrophy is a risk factor for the development of congestive heart failure, sudden death, and myocardial infarction.1 2 Pressure-overload cardiac hypertrophied hearts also have an increased susceptibility to myocardial injury after ischemia and reperfusion.3 4 5 It has been suggested that alterations in energy substrate use in the hypertrophied hearts contribute to these findings.6 7 8 9 10 11 12 13 14 15 16 Two prominent alterations in energy substrate metabolism observed in hypertrophied hearts are (1) a decrease in fatty acid oxidation rates10 12 with (2) a parallel increase in glycolytic rates.12
It is well established that tissue carnitine levels are decreased in the hypertrophied myocardium.10 12 13 14 15 16 17 Carnitine is an essential cofactor required for the translocation of activated long-chain fatty acids into the inner mitochondrial matrix. A limitation in the translocation of fatty acids into the mitochondria due to decreased carnitine levels has been suggested to lead to decreased ATP production from fatty acid oxidation and, thereby, myocardial dysfunction.10 16 For this reason, restoring myocardial carnitine levels is a potential therapeutic approach to ameliorate the pathophysiological consequences of myocardial hypertrophy. However, although fatty acid oxidation has been shown to be decreased in isolated perfused hypertrophied hearts,10 12 this decrease is dependent on the perfusion conditions used. At high workloads, the decrease in fatty acid oxidation is not as evident.12
PLC is a naturally occurring derivative of carnitine that has the potential to not only replenish myocardial carnitine stores18 but also to replenish key mitochondrial TCA intermediates.19 20 In human subjects with preexisting ventricular dysfunction, PLC administration results in an improved myocardial contractility and function.21 Several studies have also shown beneficial effects of PLC on in vivo and in vitro functional parameters of hypertrophied hearts in experimental animal preparations.13 14 15 PLC also improves the mechanical function of both normal and hypertrophied hearts after ischemia.18 22 Although the beneficial effects of PLC have been suggested to occur via improvement of fatty acid oxidation, this has yet to be directly determined.
In addition to its involvement in fatty acid oxidation, carnitine also has an important role in regulating glucose metabolism. We recently demonstrated that increasing myocardial carnitine levels stimulates glucose oxidation in the normal heart.23 24 This stimulation of glucose oxidation can be attributed to the actions of carnitine on inner mitochondrial carnitine acetyltransferase, which enhances conversion of mitochondrial acetyl CoA to cytoplasmic acetylcarnitine.25 The decrease in the ratio of mitochondrial acetyl CoA to CoA results in activation of the PDC, thereby increasing glucose oxidation rates. Hypertrophied hearts also have alterations in glucose metabolism, with elevated glycolytic enzyme activity and high rates of glycolysis compared with normal hearts.6 8 12 These alterations can exaggerate the imbalance between glucose oxidation and glycolysis12 26 and potentially have a negative impact on heart function because of an increase in hydrogen ion (H+) derived from glucose metabolism.26 27
Although it is generally assumed that PLC improves myocardial energy metabolism by increasing fatty acid oxidation, it is possible that the beneficial effects of PLC could also occur secondary to a stimulation of glucose oxidation. To date, the direct effects of PLC on overall energy substrate metabolism have not been determined. The present study was designed to directly determine the effects of PLC on myocardial carbohydrate and fatty acid metabolism in hypertrophied rat hearts. Rates of glycolysis, glucose oxidation, lactate oxidation, and fatty acid oxidation were measured in isolated working hearts pretreated with PLC. The effects of a chronic administration of PLC to aortic-banded rats on the contribution of these pathways to ATP production were also determined. Our results demonstrate that the primary effect of PLC in hypertrophied hearts is to increase carbohydrate oxidation.
Materials and Methods
d-[5-3H]Glucose, d-[U-14C]glucose, [U-14C]lactate, and [9,10-3H(N)]palmitate were purchased form 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. PLC (lot 920068) was obtained from Prassis Sigma Tau Research Institute. All other chemicals were reagent grade.
Juvenile male Wistar-Kyoto rats weighing 50 to 80 g were anesthetized with methohexital sodium (50 mg · kg−1 IP, brietal sodium). The suprarenal abdominal aorta was subsequently isolated and clipped with a minimally occlusive (0.4-mm) hemoclip.12 Control animals had the aorta isolated but not clipped.
Isolated Heart Preparation
Eight weeks after the surgery, hearts were quickly removed from pentobarbital-anesthetized (60 mg · kg−1 IP) rats and placed in ice-cold Krebs-Henseleit buffer. The aorta was immediately cannulated, and retrograde perfusion was initiated. During this initial Langendorff perfusion, the left atrium was also cannulated. Hearts were then switched to the working mode and perfused at a 11.5 mm Hg left atrial preload and 80 mm Hg aortic afterload. In experiments involving the acute administration of PLC, a modified Krebs-Henseleit buffer gassed with 95% O2/5% CO2 (pH 7.4) was used; the buffer contained 2.5 mmol/L free Ca2+, 11 mmol/L glucose, 0.5 mmol/L lactate, 1.2 mmol/L palmitate prebound to 3% bovine serum albumin, and 100 μU · mL−1 insulin. In experiments involving the chronic administration of PLC, the perfusion buffer contained 11 mmol/L glucose, 0.5 mmol/L lactate, 0.4 mmol/L palmitate prebound to 3% bovine serum albumin, and 100 μU · mL−1 insulin. In this series of perfusions, the working hearts were paced at 280 bpm, the left atrial preload was set at 11.5 mm Hg, and the aortic afterload outflow line was set at 80 mm Hg.
Left ventricular peak systolic pressure, measured with a Spectramed P23 XL pressure transducer in the aortic afterload line, was recorded on a Gould RS-3600 physiograph. Left ventricular developed pressure was determined by subtracting the peak systolic pressure from the diastolic pressure. Cardiac output and aortic flow were measured with Transonic ultrasonic flow probes present in the atrial preload line and the aortic outflow lines, respectively. Coronary flow was calculated as the difference between cardiac output and aortic flow.
Isolated working hearts were initially perfused for a 60-minute period with buffer containing 1 mmol/L PLC. Hearts not loaded with PLC were perfused for a comparable time with PLC-free buffer. At the end of this 60-minute period, hearts were switched to a Langendorff perfusion for 5 minutes to wash out any extracellular PLC. The perfusion buffer was then changed to a PLC-free buffer containing appropriate radiolabeled substrates for the measurement of substrate utilization (see below). Glycolysis, glucose oxidation, lactate oxidation, and fatty acid oxidation were measured in working hearts during a subsequent 30-minute perfusion. Metabolic samples were taken at 10-minute intervals during this period.
The effects of chronic PLC administration on energy metabolism were also determined. In the untreated group, both sham-operated and aortic-banded animals were fed food and water ad libitum for the 8-week study period. In the treated group, the control and aortic-banded animals underwent a similar protocol except that PLC was present in the drinking water throughout the 8-week period after banding. The final concentration of the drinking water was adjusted such that the animals consumed 60 mg · kg−1 · day−1 of PLC.
Measurement of Glycolysis and Glucose Oxidation
Rates of glycolysis and glucose oxidation were measured simultaneously in hearts as previously described.27 Rates of glycolysis were measured by quantitative determination of the amount of 3H2O liberated from the labeled [5-3H]glucose in the buffer. Separation of 3H2O from [3H/14C]glucose was achieved as previously described28 via the use of Dowex 1-X4 anion exchange columns (200- to 400-mesh). Dowex 1-X4 anion exchange resin (with a column volume of 0.5×0.5 cm) was suspended in 0.4 mol/L potassium tetraborate and washed extensively with H2O before use. A 0.2-mL sample of perfusion buffer was placed on the column and eluted into scintillation vials by the addition of 0.8 mL H2O to the column. The columns were found to retain 98% to 99.6% of the total [3H/14C]glucose. After addition of Ecolite scintillant to the eluant, the samples were counted by using a double-isotope β-scintillation counting procedure. The signal obtained from the 3H2O (which elutes from the column) was corrected for the small amount of [3H]glucose that also passes through the column by determining the amount of [14C]glucose eluted. The small amount of spillover from the 14C signal into the 3H window was also corrected by standards that contained only 14C.
Rates of glucose oxidation were measured by quantitatively collecting 14CO2 produced by the heart from [14C]glucose in the buffer, as described previously.27 The 14CO2 produced as a gas was determined by bubbling gas in the closed perfusion system through a 1 mol/L methylbenzethonium hydroxide 14CO2 trap. This allowed for the quantitative collection of all 14CO2 released as a gas. The hyamine hydroxide solution was sampled at 10-minute intervals during the 30-minute period in which glucose oxidation was measured. At the same time, duplicate perfusate samples were collected and stored under mineral oil to prevent the liberation of 14CO2, and the 14CO2 was subsequently extracted from the [14C]bicarbonate, as described previously.27 This involved injecting perfusate samples into closed metabolic reaction flasks containing 9N H2SO4 and gently shaking for 1 hour. The 14CO2 released from the perfusion buffer was trapped in center wells filled with 1 mol/L methylbenzethonium hydroxide. The center wells were subsequently removed and counted in ACS scintillant by using standard β-scintillation counting procedures.
Measurement of Lactate and Palmitate Oxidation
Rates of lactate and palmitate oxidation were measured in the same hearts, which were perfused under conditions identical to those for the series of hearts in which glycolysis and glucose oxidation were measured, except that the lactate and palmitate contained the radiolabel.12 Lactate oxidation rates were measured from [U-14C]lactate in the buffer by quantitatively collecting 14CO2 production in a manner similar to that described for glucose oxidation.
Palmitate oxidation rates were measured by quantitative determination of the rate of 3H2O production from [9,10-3H]palmitate, as described previously.28 Briefly, this method entails the extraction of perfusion buffer samples with a 1:2 (vol/vol) mixture of chloroform/methanol. After separation into aqueous and organic chloroform phases, the aqueous phase was taken and extracted by using a mixture of chloroform, methanol, and 2 mol/L KCl/HCl (1:1:0.9). Samples of the aqueous phase were taken and counted to determine the content of 3H2O after the addition of scintillant. This method results in a >99.7% extraction and separation of 3H2O from the [3H]palmitate.28 Spillover of [14C]lactate into the aqueous phase of the extract was taken into account.
At the end of perfusion, heart ventricles were freeze-clamped by 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 and weighed, while frozen ventricular tissue was powdered by use of a mortar and pestle cooled to the temperature of liquid nitrogen. A portion of the tissue was dried and used to calculate the dry weight–to–wet weight 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.
Myocardial carnitine content was determined by exposing a small quantity of frozen tissue (50 mg) to a period of alkaline hydrolysis (1 mL of 0.5 mmol/L KOH at 70°C for 1 hour). This results in the hydrolysis of short- and long-chain carnitine esters and the production of free carnitine. A radiometric assay was used to determine the total free carnitine content of the myocardium.29
The data are represented as the mean±SEM. When comparing two group means, Student’s t test was used. A value of P≤.05 was regarded as significant.
Characteristics of Aortic-Banded Rats
Heart and body weights in the aortic-banded and sham-operated rats are shown in Table 1⇓. At the end of the 8-week study period, a mild cardiac hypertrophy was seen in the aortic-banded rats, with heart weight increasing by 15% compared with sham-operated control rats. A significant hypertrophy was seen regardless of whether it was based on differences in heart wet weight or dry weight. The heart weight–to–body weight ratio was also significantly increased in the aortic-banded rats. Aortic banding did not significantly affect the body weight of the rats.
Myocardial carnitine content in hypertrophied and control hearts is shown in Fig 1⇓. Similar to previous studies,10 12 13 14 15 16 17 a significant decrease in myocardial carnitine levels was seen in the hypertrophied hearts. Perfusion of hearts for a 60-minute period with 1 mmol/L PLC resulted in a significant increase in carnitine levels in both the control and hypertrophied hearts. Total myocardial carnitine content increased 35% in control hearts and 47% in hypertrophied hearts.
PLC Effects on Mechanical Function in Hypertrophied Hearts
CW in hypertrophied and control hearts is shown in Fig 2⇓. The mildly hypertrophied hearts used in the present study showed no significant difference in CW from that found in control hearts at the initiation of the perfusion. The addition of 1 mmol/L PLC did not initially alter CW in either hypertrophied or control hearts. However, by 60 minutes of perfusion, a significant increase in CW was observed in the PLC-treated hypertrophied hearts compared with untreated hypertrophied hearts. A similar increase was not observed in control hearts. This improvement in CW in PLC-treated hypertrophied hearts persisted even after the washout of the PLC from the perfusate.
Table 2⇓ shows the effects of PLC on other parameters of heart function at the end of the 30-minute period following the removal of PLC from the perfusate. The main effect of PLC was to increase aortic flow in the hypertrophied hearts, resulting in a significant increase in cardiac output. No major effect of PLC was observed on either heart rate, developed pressure, peak systolic pressure, or coronary flow.
It should be noted that although heart weight in the hypertrophied hearts was 15% higher than in the control hearts, coronary flow did not differ between the two groups. Despite this, it is unlikely that the hypertrophied hearts were perfused under slightly ischemic conditions. Mechanical function was well maintained in these hearts and increased upon PLC supplementation without any further increase in coronary flow, suggesting that the O2 supply was not limited. Although we did not measure CK release in these particular hearts, we have measured CK production in a series of hypertrophied hearts perfused under similar conditions and did not see any release, and we have also observed that glycogen levels are maintained in hypertrophied hearts perfused under these conditions, which would not be expected if ischemia were present (B.O. Schönekess, M.F. Allard, and G.D. Lopaschuk, unpublished data, 1995).
PLC Effects on Energy Substrate Metabolism in Hypertrophied Hearts
After removal of PLC, steady state rates of glycolysis, glucose oxidation, lactate oxidation, and fatty acid oxidation were subsequently measured over a 30-minute period (Fig 3⇓). As shown in Fig 3A⇓, rates of glycolysis were significantly accelerated in hypertrophied hearts compared with control hearts. As expected, glucose oxidation rates (Fig 3B⇓) were considerably lower than glycolytic rates in all hearts. This is due to the fact that fatty acids are more potent inhibitors of glucose oxidation than of glycolysis (secondary to inhibition of PDC by acetyl CoA derived from β-oxidation). Although glycolytic rates were elevated in hypertrophied hearts, a significant decrease in glucose oxidation rates was seen in these hearts compared with control hearts (Fig 3B⇓).
In hearts treated with PLC, there was a dramatic increase in glucose oxidation rates in both the control and hypertrophied hearts (Fig 3B⇑). Rates of glucose oxidation increased from 216.6±26.1 to 857.7±192.4 nmol · min−1 · g dry wt−1 in control hearts and from 136.7±24.5 to 626.7±109.7 nmol · min−1 · g dry wt−1 in hypertrophied hearts. The increase in glucose oxidation was also accompanied by a significant increase in glycolysis in control hearts (from 2320±394 to 4183±768 nmol · min−1 · g dry wt−1). Rates of glycolysis were not further increased in hypertrophied hearts (from 3792±352 to 4467±958 nmol · min−1 · g dry wt−1).
Rates of lactate oxidation in hypertrophied and control hearts are shown in Fig 3C⇑. Similar to glucose oxidation rates, lactate oxidation rates were significantly depressed in the hypertrophied hearts compared with control hearts (118.7±17.1 versus 194.4±25.2 nmol ·min−1 · g dry wt−1, respectively). PLC treatment significantly increased the rate of lactate oxidation in hypertrophied hearts (from 118.7±17.1 to 252.2±47.4 nmol · min−1 · g dry wt−1). In contrast, lactate oxidation rates were not significantly increased in control hearts treated with PLC (194.4±25.2 vs 253.7±62.3 nmol ·min−1 · g dry wt−1).
Rates of palmitate oxidation in hypertrophied and control hearts are shown in Fig 3D⇑. No differences in steady state rates of palmitate oxidation were seen in either group. This differs from hearts perfused with low concentrations of palmitate (0.4 mmol/L), in which we previously observed a significant decrease in fatty acid oxidation in hypertrophied hearts compared with control hearts.12 PLC treatment significantly increased palmitate oxidation in hypertrophied hearts (from 379.6±32.1 to 529.9±56.7 nmol · min−1 · g dry wt−1) but not in PLC-treated control hearts (429.1±59.1 versus 394.9±34.7 nmol · min−1 · g dry wt−1).
Rates of oxidative metabolism in the heart are very dependent on the actual work performed by the heart.30 Since CW varied considerably between the experimental groups, we also normalized the rates of glucose and palmitate oxidation for CW. The significant increase in glucose oxidation after PLC treatment remained when normalized for CW in both control hearts (from 1.77±0.25 to 3.68±0.89 nmol · min−1 · CW−1, P≤.05) and hypertrophied hearts (from 1.50±0.34 to 3.53±1.05 nmol · min−1 · CW−1, P≤.05). In contrast, rates of palmitate oxidation normalized for CW do not significantly increase upon acute loading of PLC in the control group (2.38±0.23 versus 2.77±0.22 nmol · min−1 · CW−1) or the hypertrophied group (2.21±0.13 versus 2.52±0.30 nmol · min−1 · CW−1).
Steady State Rates of ATP Production in Hypertrophied Hearts
Overall rates of ATP produced from exogenous carbon substrates were calculated from the steady state glycolytic and oxidative rates obtained in Fig 3⇑. For glycolysis, the net production of ATP is 2 mol per mole of glucose passing through the glycolytic pathway. When glucose is oxidized, 36 mol of ATP are produced per mole of glucose (this does not include the 2 mol of ATP from glycolysis). Lactate produces 18 mol of ATP per mole of lactate, and the oxidation of palmitate produces 129 mol of ATP per mole of palmitate. Absolute rates of ATP production in control and hypertrophied hearts are shown in Fig 4A⇓. Similar rates of overall ATP production were observed in control and hypertrophied rat hearts perfused in the absence of PLC. PLC treatment increased rates of ATP production in both control and hypertrophied hearts. This increase was mainly due to effects of PLC on carbohydrate oxidation.
Fig 4B⇑ shows the percent contribution of glycolysis, glucose oxidation, lactate oxidation, and fatty acid oxidation to ATP production. The most significant finding is that in the absence of PLC, control hearts derived 7% of ATP production from glycolysis compared with 12% in hypertrophied hearts. The contribution of palmitate oxidation to ATP production was 77% in hypertrophied hearts compared with 80% in control hearts. In PLC-treated hearts, a dramatic increase in the relative contribution of glucose oxidation to ATP production occurred in both hypertrophied hearts (from 8% to 22%) and control hearts (from 8% to 33%). The relative contribution of lactate to ATP production did not change dramatically in any of the experimental groups. In the PLC-treated hypertrophied hearts, the increase in absolute rates of palmitate oxidation seen in Fig 3D⇑ did not translate into a relative increase in palmitate oxidation to ATP production. This is because the increase in palmitate oxidation paralleled the increase in mechanical function and overall ATP production seen in these hearts (Fig 4B⇑).
Effects of Chronic PLC Administration to Aortic-Banded Animals on ATP Production Rates
We also determined whether the in vivo treatment of animals with PLC had an effect on energy substrate preference similar to that found after the acute administration of PLC. In this series of experiments, aortic banding resulted in an 18.4% (P<.05) increase in heart weight (wet heart weight was 1.501±0.028 g [n=18] and 1.777±0.074 g [n=22] in control and aortic-banded rats, respectively). Treatment with PLC did not significantly alter heart weight in either control or aortic-banded rats (1.459±0.043 g [n=26] and 1.907±0.083 g [n=26], respectively). This chronic treatment protocol resulted in a significant increase in myocardial carnitine content in both the control hearts (5465±474 and 7156±276 nmol · g dry wt−1 in untreated and treated animals, respectively; P<.05) and hypertrophied hearts (4526±197 and 6411±305 nmol · g dry wt−1 in untreated and treated animals, respectively; P<.05).
Hearts from these animals were subsequently perfused, in the absence of added PLC, with low levels of fatty acids (0.4 mmol/L palmitate). No difference in CW was observed in control hearts obtained from untreated or PLC-treated animals (CW was 39.4±3.8 [n=16] and 41.9±3.5 [n=17] mm Hg · mL · min−1 · 10−2, respectively). In hypertrophied hearts from untreated animals, a significant decrease in CW was observed compared with untreated control hearts (26.5±3.3 [n=18] versus 39.4±3.8 [n=16] mm Hg · mL · min−1 · 10−2, respectively; P<.05). In hypertrophied hearts treated with PLC, a significant improvement in CW was observed compared with hypertrophied hearts from untreated animals (36.0±3.7 [n=21] versus 26.5±3.3 [n=17] mm Hg · mL · min−1 · 10−2, respectively; P<.05).
The effects of chronic in vivo PLC treatment on the percent contribution to ATP production of glycolysis, glucose oxidation, lactate oxidation, and palmitate oxidation are shown in Fig 5⇓. As expected, in control and hypertrophied hearts, glucose oxidation provides a greater proportion of ATP production if hearts are perfused with low concentrations of fatty acids (0.4 mmol/L palmitate). In control hearts, glucose oxidation provided 36% of ATP production compared with the 8% contribution in hearts perfused with 1.2 mmol/L palmitate (see Fig 4⇑). In hypertrophied hearts, glucose oxidation also provided the majority of ATP production (41%), with palmitate oxidation providing 34%. Chronic treatment with PLC had little effect on the contribution of glycolysis, glucose oxidation, lactate oxidation, and palmitate oxidation in control hearts. In contrast, PLC treatment increased the contribution of glucose oxidation to 46% in hypertrophied hearts and decreased the contribution of fatty acid oxidation to 30%. Steady state rates of palmitate oxidation were not affected by chronic PLC treatment in the hypertrophied heart. Rates of palmitate oxidation were 170±24 nmol · min−1 · g dry wt−1 in untreated hypertrophied hearts compared with 145±25 nmol · min−1 · g dry wt−1 in PLC-treated hearts. This suggests that the improvement of CW in hypertrophied hearts after chronic treatment of PLC could not be accounted for by an increase in palmitate oxidation.
A number of studies have demonstrated that PLC can improve contractile function in hypertrophied hearts.13 14 15 This has been attributed, in part, to the ability of PLC to replenish myocardial carnitine stores, which are significantly depressed in the hypertrophied heart.10 12 13 14 15 16 17 Decreased myocardial carnitine has been suggested to be responsible for the depression of fatty acid oxidation observed in the hypertrophied heart.10 15 In the present study, we also observed a decrease in myocardial carnitine content, even though hearts were only mildly hypertrophied. However, despite the fact that the decrease in carnitine was of the same magnitude as what has been observed in more severely hypertrophied hearts,10 we did not see any decrease in fatty acid oxidation rates. Rather, the main alterations in energy substrate metabolism of hypertrophied hearts were an increase in glycolysis and a decrease in carbohydrate oxidation (glucose and lactate) compared with control hearts. Acute PLC treatment was effective in completely reversing the decrease in carnitine seen in the hypertrophied hearts. Interestingly, the primary effect of PLC treatment was not to increase fatty acid oxidation but to increase carbohydrate oxidation. This increase in carbohydrate oxidation was accompanied by an increase in overall ATP production and an increase in contractile function in the hypertrophied hearts. It is interesting to find that when rates of glucose and palmitate oxidation are normalized for the increased CW performed by the hypertrophied heart, PLC treatment did not increase fatty acid oxidation but increased carbohydrate oxidation. Chronic administration of PLC (60 mg · kg−1 · day−1) was also effective in increasing myocardial carnitine content and improving cardiac function in hypertrophied hearts. In these hearts, the primary effect of PLC was to increase the relative contribution of glucose oxidation to ATP production and to decrease the relative contribution of palmitate oxidation to ATP production without altering steady state rates of fatty acid oxidation. These observations suggest that the beneficial effects of PLC in hypertrophied hearts are primarily the result of its ability to stimulate carbohydrate oxidation.
Carnitine is an important cofactor necessary for the oxidation of fatty acids. A key enzyme involved in fatty acyl CoA uptake by the mitochondria, CPT 1, has an absolute requirement for carnitine. For this reason, a decrease in tissue carnitine has the potential to decrease fatty acid oxidation by limiting acyl CoA transport into the mitochondria. Recently, carnitine has also been recognized as having an important role in regulating carbohydrate oxidation.23 24 This is thought to occur secondary to a stimulation of inner mitochondrial carnitine acetyltransferase.25 This enzyme transfers acetyl groups from mitochondrial acetyl CoA to cytoplasmic acetylcarnitine, resulting in a decrease in the inner mitochondrial acetyl CoA–to–CoA ratio. A decrease in this ratio will stimulate the PDC, the enzyme that converts pyruvate to acetyl CoA, and is the rate limiting step of glucose oxidation. In support of this conclusion, we have previously demonstrated that increasing carnitine levels in hearts will increase the rate of glucose oxidation in fatty acid-perfused hearts.23 As shown in Fig 3B⇑, PLC was also effective in stimulating glucose oxidation in both control and hypertrophied hearts. This probably occurred secondary to its effect of increasing carnitine levels and stimulating PDC in these hearts. As a result, a greater proportion of the pyruvate derived from glycolysis (glucose oxidation) as well as pyruvate derived from lactate (lactate oxidation) was oxidized.
The effects of PLC on energy metabolism may also have occurred secondary to replenishing TCA cycle intermediates, thereby increasing overall TCA cycle activity. PLC is an effective anaplerotic substrate,19 20 with the propionyl group being used to synthesize succinyl CoA. PLC did increase overall ATP production rates (and therefore overall TCA cycle activity) in the hypertrophied hearts. Whether the increase in TCA cycle activity was responsible for the increase in CW or vice versa cannot be unequivocally concluded from the present study.
The relation between myocardial carnitine levels and either fatty acid oxidation or carbohydrate oxidation has not been clearly delineated. In severe carnitine deficiencies, a depression in fatty acid oxidation occurs that can compromise muscle function. Treatment of rats with Na+ pivalate also results in a marked decrease in myocardial carnitine content, with a parallel decrease in fatty acid oxidation rates.30 Although it is clear that tissue levels of carnitine are decreased in the hypertrophied heart, it is not clear whether this is responsible for a decrease in fatty acid oxidation. In previous studies, El Alaoui-Talibi et al10 and our group12 found that fatty acid oxidation rates can be depressed in isolated working hearts from aortic-banded rats. However, in our previous studies, the reduction of fatty acid oxidation was most obvious at low workloads in the presence of low concentrations of fatty acids.12 Fatty acid oxidation was not reduced in hypertrophied hearts subjected to increasing CW12 or in the presence of higher concentrations of fatty acids (Fig 3D⇑). Thus, it does not appear that decreased fatty acid oxidation rates are limiting contractile function in the mildly hypertrophied heart. However, this may not be true in severely hypertrophied hearts; Cheikh et al16 have shown that supplying these hearts with a carbon substrate that bypasses CPT 1 (octanoate) improves their energetics.
Our findings suggest that stimulation of carbohydrate oxidation is the primary metabolic effect of carnitine supplementation either in normal hearts23 or in the carnitine deficiency seen in hypertrophied hearts (Fig 3B⇑ and 3C⇑). In hearts perfused with high levels of palmitate, a significant decrease in both glucose and lactate oxidation was observed in the hypertrophied hearts compared with the control hearts. Replenishing myocardial carnitine levels with acute PLC treatment not only reversed this decrease but also resulted in a further increase in rates of carbohydrate oxidation. PLC treatment markedly increased overall ATP production and the contribution of carbohydrate oxidation to ATP production. In light of the last findings, we postulate that in hearts with mild carnitine deficiency, a decrease in carbohydrate oxidation occurs, secondary to a decrease in carnitine acetyltransferase activity. A limitation of CPT 1 activity and fatty acid oxidation would not likely occur until the carnitine deficiency was more severe. Paradoxically, we anticipate that carnitine deficiencies severe enough to inhibit fatty acid oxidation may actually increase carbohydrate oxidation. In this setting, the decrease in the mitochondrial acetyl CoA–to–CoA ratio that would occur secondary to a decrease in β-oxidation would relieve the inhibition of PDC. In support of this concept, it has been demonstrated that the severe carnitine deficiency that occurs in hearts of Na+ pivalate–fed rats is accompanied by an decrease in fatty acid oxidation and an increase in glucose oxidation.31
In the present study, we observed a significant increase in glycolysis in hypertrophied hearts compared with control hearts. This increase in glycolysis, which is also seen in hypertrophied hearts perfused with low concentrations of fatty acids,12 is consistent with the increase in enzyme activities associated with glycolysis.6 8 Despite this increase in glycolysis, rates of glucose oxidation were decreased in hypertrophied hearts compared with normal hearts. These metabolic alterations create a dramatic uncoupling of glycolysis from glucose oxidation, resulting in a greater amount of pyruvate derived from glycolysis being converted to lactate. Uncoupling of glycolysis from glucose oxidation is a major source of H+ production in the heart.27 32 Hydrolysis of ATP from each molecule of glucose that passes through glycolysis but not glucose oxidation produces two H+ molecules. In contrast, glycolysis coupled to glucose oxidation is a H+-neutral process.32 The excess production of H+ due to an exaggerated uncoupling of glycolysis from glucose oxidation may contribute to contractile dysfunction in the hypertrophied heart. Recently, Hata et al33 demonstrated that H+ accumulation in the myocardium can markedly decrease cardiac efficiency. They postulate that this most likely occurs secondary to a stimulation of Na+-H+ exchange activity, with a subsequent increase in Na+-Ca2+ exchange activity. The accumulation of Na+ and Ca2+ that occurs secondary to clearance of H+ requires that a greater amount of ATP be directed toward basal metabolism, as opposed to contractile function. If the same situation is occurring in the hypertrophied heart because of an uncoupling of glycolysis and glucose oxidation, then stimulation of glucose oxidation may partly explain the benefit of PLC. In this regard, we have recently demonstrated that cardiac efficiency is improved by chronic PLC administration to aortic-banded rats (B.O. Schönekess, M.F. Allard, and G.D. Lopaschuk, unpublished data, 1995). As a result, PLC has the potential not only to increase overall ATP production but also to improve the efficiency of translating this ATP into contractile function.
In summary, we have shown that PLC treatment (both acute and chronic) significantly increases CW in mildly hypertrophied hearts. The benefits of PLC treatment are not related to an increase in the contribution of fatty acid oxidation per unit work or to PLC as a source of ATP production in the hypertrophied heart. Rather, the primary effect of PLC is to increase the contribution of carbohydrate oxidation to ATP production, presumably as a consequence of a reduction in the mitochondrial acetyl CoA–to–CoA ratio brought about by stimulation of carnitine acetyltransferase. This suggests that inner mitochondrial carnitine acetyltransferase may be an important target for improving energy substrate metabolism in the hypertrophied heart.
Selected Abbreviations and Acronyms
|PDC||=||pyruvate dehydrogenase complex|
This study was supported by Prassis Sigma Tau Research Institute, Milano, Italy, and 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. Mr Schönekess is a graduate student trainee of the Alberta Heritage Foundation for Medical Research and 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 December 2, 1994.
- Accepted June 6, 1995.
- © 1995 American Heart Association, Inc.
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