Decreased Contractile and Metabolic Reserve in Peroxisome Proliferator–Activated Receptor-–Null Hearts Can Be Rescued by Increasing Glucose
http://www.100md.com
《循环学杂志》
the NMR Laboratory for Physiological Chemistry, Division of Cardiovascular Medicine, Brigham and Women’s Hospital, Boston, Mass (I.L., J.A.B., Y.X., R.T.), and Center for Cardiovascular Research, Washington University School of Medicine, St Louis, Mo (D.P.K., T.C.L.).
Abstract
Background— Downregulation of peroxisome proliferator–activated receptor- (PPAR) in hypertrophied and failing hearts leads to the reappearance of the fetal metabolic pattern, ie, decreased fatty acid oxidation and increased reliance on carbohydrates. Here, we sought to elucidate the functional significance of this shift in substrate preference.
Methods and Results— We assessed contractile function and substrate utilization using 13C nuclear magnetic resonance spectroscopy and high-energy phosphate metabolism using 31P nuclear magnetic resonance spectroscopy in perfused hearts isolated from genetically modified mice (PPAR–/–) that mimic the metabolic profile in myocardial hypertrophy. We found that the substrate switch from fatty acid to glucose (3-fold down) and lactate (3-fold up) in PPAR–/– hearts was sufficient for sustaining normal energy metabolism and contractile function at baseline but depleted the metabolic reserve for supporting high workload. Decreased ATP synthesis (measured by 31P magnetization transfer) during high workload challenge resulted in progressive depletion of high-energy phosphate content and failure to sustain high contractile performance. Interestingly, the metabolic and functional defects in PPAR–/– hearts could be corrected by overexpressing the insulin-independent glucose transporter GLUT1, which increased the capacity for glucose utilization beyond the intrinsic response to PPAR deficiency.
Conclusions— These findings demonstrate that metabolic remodeling in hearts deficient in PPAR increases the susceptibility to functional deterioration during hemodynamic overload. Moreover, our results suggest that normalization of myocardial energetics by further enhancing myocardial glucose utilization is an effective strategy for preventing the progression of cardiac dysfunction in hearts with impaired PPAR activity such as hearts with pathological hypertrophy.
Key Words: fatty acids glucose metabolism myocardium
Introduction
Peroxisome proliferator–activated receptor (PPAR), a nuclear receptor that regulates the expression of multiple genes controlling both fatty acid uptake and oxidation, is a key player in the transcriptional regulation of substrate preference in the heart.1–7 Downregulation of PPAR, widely observed in diseased hearts such as hearts with cardiac hypertrophy and failure, allows shifting substrate preference from fatty acid to glucose.8 It is not clear whether decreased PPAR activity in hypertrophied hearts is adaptive or maladaptive. Although some studies have suggested that PPAR deficiency renders a fetal metabolic profile and hence impaired functional capacity of the heart, others have shown that reactivation of PPAR worsens contractile function in the hypertrophied and failing heart, suggesting that the downregulation of PPAR is a necessary adaptation.2,3,5,9
Clinical Perspective p 2346
In this study, we sought to determine the functional significance of PPAR deficiency by analyzing cardiac substrate metabolism, myocardial energetics, and contractile function in PPAR–/– hearts. Our findings show that PPAR–/– hearts are able to maintain baseline function but manifest abnormal myocardial energetics, inefficient ATP generation, and impaired contractile function at high workload. These results suggest that a compensatory increase in carbohydrate utilization in response to the downregulation of PPAR occurs at the expense of depleting the metabolic reserve of the heart. Furthermore, by overexpressing a glucose transporter in PPAR–/– hearts, we demonstrate that the inadequate substrate switch can be improved by upregulating the capacity for glucose utilization.
Methods
Animal Models
The PPAR–/– mice (kindly provided by Dr Frank Gonzalez) have been described previously.10 PPAR–/–TG mice were generated by crossing a mouse that overexpresses the insulin-independent glucose transporter GLUT1 (TG) in a cardiac-specific manner11 with PPAR–/– mice to enhance glucose transport in the hearts of PPAR–/– mice. PPAR–/– mice and GLUT1-TG mice (on sv129 and FVB background, respectively) were crossed to generate PPAR+/– and PPAR+/–TG mice. These heterozygous mice were used as breeders. Among the 6 resulting genotypes, PPAR–/–, PPAR–/–TG, TG, and wild type (WT) (1/8 for each genotype expected in every litter) were used for the experiments, and PPAR+/– and PPAR+/–TG (1/4 for each genotype expected in every litter) were used as new breeders.
Isolated Perfused Heart Experiments and 31P Nuclear Magnetic Resonance Spectroscopy
Mice were given heparin (100 U IP) and anesthetized by sodium pentobarbital (150 mg/kg IP). The heart was excised and perfused at a constant pressure of 80 mm Hg at 37°C as previously described.12 The perfusate contains the following (in mmol/L): NaCl 118, NaHCO3 25, KCl 5.3, CaCl2 2, MgSO4 1.2, EDTA 0.5, glucose 5.5, mixed long-chain fatty acids 0.4 bound to 1% albumin, DL--hydroxybutyrate 0.38, lactate 2, and insulin 50 μU/mL equilibrated with 95% O2 and 5% CO2 (pH 7.4). A water-filled balloon was inserted into the left ventricle to record ventricular pressure and heart rate. Balloon volume was adjusted to achieve an end-diastolic pressure of 5 to 10 mm Hg. Isovolumic contractile function was estimated by the product of left ventricular (LV) developed pressure and heart rate (rate-pressure product; RPP). Myocardial oxygen consumption (MVO2) was determined by measuring the coronary flow rate and the PO2 difference between perfusate and effluent from the pulmonary outflow tract.13
After a 30-minute equilibration period, baseline function was recorded, and the perfusion medium was switched to a buffer containing 13C-enriched substrates. To elicit a high contractile performance, concentration of CaCl2 in the perfusate was increased from 2 to 4 mmol/L. Hearts were maintained at either baseline or high workload level for 25 minutes. At the end of the experiments, hearts were freeze clamped with Wollenberger tongs precooled in liquid nitrogen.
31P nuclear magnetic resonance (NMR) spectroscopy of isolated perfused mouse hearts was performed as previously described.12 One 8-minute spectrum was collected at baseline, and 3 consecutive spectra were collected during high workload. In separate hearts, ATP synthesis rates were measured with the 2-site saturation transfer technique by applying a low-power narrow-band radiofrequency pulse to saturate -ATP resonance and observing changes in the Pi resonance area.14,15 Spectra were acquired before (M0)and 4.8 seconds after (M) the saturating pulse was applied by signal averaging 256 total scans as interleaved sets of 8 scans. A complete measurement was acquired in 46 minutes.
13C NMR Spectroscopy
Perchloric acid extracts were prepared from freeze-clamped heart tissue, neutralized by KOH, lyophilized, and subsequently dissolved in 300 μL D2O. Proton-decoupled 13C NMR spectra of tissue extracts were obtained with a Varian Inova 400 spectrometer 3-mm NMR probe. Data were collected using a 60° pulse with a 3.0-second delay, a sweep width of 10 000 Hz, and 16 000 data points. The contributions of each labeled substrate and the total of the unlabeled exogenous and endogenous substrates to the oxidative metabolism were determined using the peak areas of 13C isotopomers at C3 and C4 of glutamate by modeling the tricarboxylic acid (TCA) cycle fluxes as previously described.16,17 To distinguish between utilization of the 5 different substrate groups (glucose, fatty acids, lactate, ketones, and endogenous substrates), hearts were supplied with all substrates, 2 of which were 13C enriched for each perfusion study. In 1 series, [U-13C]fatty acid and [3-13C]lactate were used to determine the relative contributions of fatty acid and lactate. Identical experiments with enriched [U-13C]glucose and [2,4-13C]-hydroxybutyrate determined the relative contribution of these 2 substrates. The contribution from endogenous substrates was determined as the difference from 100%.
High-Performance Liquid Chromatography Measurements and Calibration of 31P NMR Spectra
Freeze-clamped tissues were used to determine the myocardial content of ATP by the high-performance liquid chromatography method as reported previously.18 Myocardial ATP content obtained by high-performance liquid chromatography was converted to [ATP] assuming an intracellular water content of 0.48 mL/g and a protein content of 0.15g/g blotted wet tissue.19 The mean values of [ATP] in hearts freeze clamped at the end of equilibration period were not different among the groups (in mmol/L) (mean±SE; n=4): WT, 11.5±0.8; PPAR–/–, 10.7±0.6; GLUT1-TG, 12.2±1.4; and PPAR–/–TG, 11.9±0.5. These values were used to calibrate ATP peak areas in baseline 31P NMR spectra for the respective groups. The area of the -ATP peak obtained under baseline conditions was set to 100% and used as the reference value for all peaks in all 31P NMR spectra.
Enzyme Activity and Glycogen Measurements
Enzyme activities of phosphofructokinase (PFK),20 GAPDH,21 lactate dehydrogenase (LDH),22 mitochondrial enzyme citrate synthase (CS),23 and cytochrome c oxidase (COX)24 were measured using freeze-clamped heart tissue as previously described. Myocardial glycogen content was determined by measuring the amount of glucose released from glycogen with alkaline extraction used to separate glycogen and exogenous glucose.19 Glucose content in the extract was measured with a Cobas Mira chemistry analyzer (Roche Applied Science). All reagents were purchased from Sigma Chemical Co and were at least analytical grade.
Statistical Analysis
All data are presented as mean±SEM. One-way ANOVA with the Bonferroni post test was performed, and an unpaired t test was used for 2-group comparisons. Analyses were performed with GraphPad Prism version 4.00 for Windows (GraphPad Software). A value of P<0.05 was considered significant.
Results
General Characteristics of the Mice
A total of 173 mice were used for this study; the average age of the animals was 29±1 weeks. Heart weight, body weight, and heart weight normalized to body weight or to tibia length were not different among the groups (Table 1). The volume of the LV balloon was similar when baseline LV end-diastolic pressure was achieved for all hearts (Table 1). Thus, there was no gross cardiac hypertrophy or dilatation in any of the groups.
Substrate Utilization Profile
The relative contributions of carbon fuel substrates to the oxidative metabolism in the heart were measured during baseline and high workload periods (Figure 1). At baseline, the contribution of fatty acids to oxidative metabolism was reduced 3-fold in PPAR–/– hearts compared with WT hearts, and the oxidation of glucose and lactate increased by 3-fold. A similar pattern was seen in PPAR–/–TG hearts at baseline. Increasing GLUT1 expression alone (TG group) led to increased glucose oxidation and decreased fatty acid oxidation, albeit less pronounced than in the PPAR–/– group. The relative contributions from -hydroxybutyrate (ketone) and endogenous substrates (glycogen and triglyceride) were not different.
In WT hearts challenged with high workload, contributions from carbohydrate (glucose and lactate) and ketone increased; the relative oxidation of exogenous fatty acids was unchanged, whereas the contribution from endogenous substrates tended to decrease. In contrast, when PPAR–/– hearts were challenged with high workload, there was no change in the substrate utilization profile compared with baseline, suggesting a loss of metabolic reserve for increasing carbohydrate utilization in these hearts. In PPAR–/–TG hearts, however, glucose oxidation at high workload increased further (up by 50% relative to PPAR–/– at high workload). Thus, increasing glucose transport in PPAR–/– hearts led to a further increase in glucose utilization beyond its intrinsic capacity and partially restored the substrate utilization response to high workload.
Glycogen Concentration and Glycolytic Enzymes
Activities of 3 major glycolytic enzymes, PFK, GAPDH, and LDH, were measured in tissue homogenates of hearts from all groups (Table 2). Despite large differences in glucose oxidation rates, the Vmax of the glycolytic enzymes was not different among the groups. This is consistent with prior observations that parallel changes in flux and enzymatic activity are not obligatory for the glycolytic pathway because the Vmax for glycolytic enzymes far exceeds the flux (>20 times) measured under most circumstances.4,25,26 Indeed, we have previously observed substantially higher glycolytic activity in TG hearts even though the Vmax was unchanged.11
Contractile Performance
We evaluated isovolumic contractile function of all hearts at 2 levels of performance. At the baseline level, all hearts were able to achieve and sustain an RPP of 35 000 mm Hg/min (Figure 3). When switched to a high-performance level, the RPP increased 2-fold in WT hearts and was sustained for the entire 25-minute period. In contrast, PPAR–/– hearts failed to maintain the high contractile performance. At the end of the high-workload period, their RPP was 25% lower than the RPP of the WT hearts (Figure 3). Importantly, contractile function during high workload was normalized in PPAR–/–TG. The contractile function of TG hearts was not different compared with the WT hearts. Decreased fatty acid oxidation in PPAR–/– hearts, despite marked increases in the oxidation of glucose and lactate, was accompanied by the inability to sustain high contractile performance, ie, a loss of contractile reserve. Interestingly, this loss was restored by further enhancing glucose transport and utilization.
Myocardial High-Energy Phosphate Content
To determine whether the altered substrate utilization observed in PPAR–/– hearts impaired high-energy phosphate metabolism leading to contractile dysfunction, we measured high-energy phosphate content and ATP synthesis rate in isolated perfused hearts. The myocardial ATP concentration was not different at baseline for WT and PPAR–/– (11.5±0.8 and 10.7±0.6 mmol/L, respectively; P=NS). During high workload, [ATP] remained fairly constant in the WT group but deteriorated progressively in the PPAR–/– group (Figure 4). The concentration of phosphocreatine (PCr), the energy reserve compound, was lower in the PPAR–/– group at baseline, reflecting a decreased energy reserve. Moreover, despite a reduced contractile performance, [PCr] remained lower during the high workload in PPAR–/– compared with WT hearts. Inorganic phosphate concentration (Pi) increased in all groups at the beginning of the high workload. However, at the end of the protocol, this increase was significantly greater in the PPAR–/– group, reflecting pronounced ATP depletion (Figure 4). The excessive depletion of high-energy phosphate content was corrected in PPAR–/–TG hearts, in which concentrations of both PCr and ATP were restored to the WT level during the high workload (Figure 4). The high-energy phosphate and inorganic phosphate contents of TG hearts were not different compared with the WT hearts. Thus, further enhancing the contribution of glucose to ATP synthesis normalized myocardial energetics in PPAR–/– hearts at high workload.
Oxygen Consumption and ATP Synthesis
The decrease in high-energy phosphate content in PPAR–/– hearts during high contractile performance suggests a defect in the balance of energy supply and consumption. Thus, MVO2 was measured at both levels of workload (Figure 5). MVO2 was similar in all groups at baseline and increased markedly at high workload. Surprisingly, the increase in MVO2 was the greatest in PPAR–/– hearts despite the least increase in contractile performance among the groups. As a result, PPAR–/– hearts had a significantly lower O2 efficiency at high workload (Figure 5). Thus, we measured ATP synthesis rate by an MVO2-independent approach using 31P NMR magnetization transfer technique. Figure 6 shows representative 31P magnetization transfer spectra obtained from a WT heart during high workload. The rate constant for ADP+PiATP was lower in PPAR–/– hearts but was restored in PPAR–/–TG hearts (0.15±0.02, 0.28±0.04, and 0.26±0.02 seconds–1 for PPAR–/–, WT, and PPAR–/–TG hearts, respectively; P<0.05, PPAR–/– versus WT). Using the flux of the reaction, representing the ATP synthesis rate, and MVO2, we calculated the P/O ratio for all groups at high workload (Table 3). Although the P/O ratio in WT is consistent with the reported value in the heart, the ratio was decreased by >50%, suggesting substantial mitochondrial uncoupling in PPAR–/– hearts.27 Oxygen consumption, ATP synthesis, and P/O ratio were not different in PPAR–/–TG and TG groups compared with the WT group. The activities of CS and COX, key enzymes for the TCA cycle and mitochondrial respiratory chain, respectively, were measured. Although CS activity was unaltered, COX activity was increased in the PPAR–/– group (Table 2), suggesting a higher O2 consumption capacity in these hearts.
Taken together, these results suggest that the ATP synthesis rate was lower in the PPAR–/– group at high workload and that MVO2 was paradoxically higher. Intriguingly, the O2 wasting was observed only when the energy demand was substantially raised by driving the heart for maximal performance. Furthermore, these defects could be normalized by increasing glucose utilization, which enabled the PPAR–/–TG hearts to sustain normal myocardial energetics despite decreased fatty acid oxidation.
Discussion
In this study, we showed that the compensatory substrate switch from fatty acids to glucose and lactate in PPAR–/– hearts was not sufficient for maintaining the high-energy phosphate content during high workload. Decreased ATP synthesis during the high workload is likely responsible for the energetic and contractile failure of PPAR–/– hearts. These results suggest that adult hearts with decreased capacity for fatty acid oxidation are more susceptible to progressive deterioration of cardiac function during hemodynamic overload. An important finding of this study is that normalization of high-energy phosphate content by further enhancing glucose utilization is sufficient to rescue these defects.
Decreased PPAR expression and activity contribute to the reappearance of the fetal metabolic pattern, ie, increased reliance on carbohydrates in cardiac hypertrophy and failure.2,3,5,28,29 It has been speculated that this switch in substrate preference may limit the functional capacity of the heart.5 Results from the present study show that hearts lacking PPAR are able to maintain the baseline function, and no gross morphological pathology is found in sedentary mice up to 30 weeks of age. However, myocardial energetics and contractile reserve were reduced in PPAR–/– hearts during high workload challenge, consistent with previous observations that these hearts were unable to cope with stresses.7,30 In this study, we tested the hypothesis that expanding the capacity for glucose utilization is sufficient to compensate for the impaired fatty acid oxidation during high energy demand. Our results support this hypothesis because further increasing glucose transport and utilization has completely rescued the energetic and contractile phenotype of PPAR–/– hearts. Furthermore, our present findings also show that increased reliance on carbohydrates per se is not harmful for the heart. However, a compensatory increase in carbohydrate utilization in PPAR–/– hearts, although sufficient to maintain energy supply for baseline work, exhausted the intrinsic reserve for any further increase in substrate utilization. The inability to increase the contribution of either glucose or fatty acids to oxidative metabolism during high energy demand renders energetic and contractile failure in PPAR–/– hearts subjected to high workload. By overexpressing GLUT1, we were able to enhance glucose utilization and to rescue the heart by 2 mechanisms: (1) increasing glucose uptake, glycolysis, and the contribution of glucose to the TCA flux to a level beyond what can be achieved in nontransgenic hearts and (2) providing more glycogen, a critical substrate for supporting the acute increase in workload.31
31P NMR saturation transfer measurements in this study showed that the ATP synthesis rate of PPAR–/– hearts was 50% lower than WT at high workload. This finding confirms that decreased ATP synthesis is responsible for the progressive loss of ATP and contractile dysfunction of these hearts at high workload. Decreased ATP synthesis at high workload has been observed previously in hypertrophied rat hearts, a model in which PPAR is downregulated.4 Our results show that decreased ATP synthesis can be rescued by further increasing the contribution of glucose to ATP synthesis. These results collectively support the notion that impaired capacity for substrate utilization leads to reduced ATP synthesis in PPAR–/– hearts at high workload. The unexpected finding in the present study is that PPAR–/– hearts waste O2 at high workload. We propose that the oxygen wasting observed here is a consequence of an energy deficit in hearts with impaired capacity for substrate utilization. This hypothesis is based on the following evidence. First, normalizing myocardial energetics by increasing glucose utilization corrected the high MVO2 in PPAR–/– hearts. Second, paradoxically high MVO2 occurs only during high energy demand, unlikely attributable to a constitutive mitochondrial defect. Although our study does not allow further elucidation of the direct causes for oxygen wasting, mitochondrial uncoupling at high workload, as revealed in the present study, will worsen the myocardial energy supply in PPAR–/– hearts and will likely contribute to the eventual contractile failure in PPAR-deficient hearts.
These observations have important clinical implications because downregulation of PPAR has been proposed as a major mechanism for substrate shift in cardiac hypertrophy and failure. Consistent with the notion that substrate switch caused by decreased PPAR activity depletes metabolic reserve, prior studies have shown that cardiac hypertrophy is associated with increased glucose utilization at baseline, but glycolysis fails to increase further during high workload. In addition, overall ATP production from exogenous fuels is decreased.1,4 Here, using transgenic mouse models as a proof-of-concept approach, we demonstrate that metabolic interventions capable of further enhancing glucose utilization have the potential to prevent the vicious cycle of progressive deterioration in pathological cardiac hypertrophy. This concept is also supported by recent studies showing that enhancing glucose utilization by either insulin-dependent or -independent mechanisms improves cardiac function and delays the transition from cardiac hypertrophy to failure.11,32 It needs to be recognized that the genetically modified mice studied here may exhibit other changes in cellular metabolism not defined by the present study. Whether these potential changes contribute to our observations is unknown. Nevertheless, because reactivation of PPAR has been found to cause contractile dysfunction in hypertrophied hearts despite the successful restoration of substrate preference profile, results from this study provide an important alternative strategy to rescue the maladaptive aspect of PPAR deficiency.
In summary, the present study shows that PPAR deficiency reduces the capacity for energy production and cardiac function in response to hemodynamic stress. Furthermore, our findings suggest that the maladaptation to the PPAR deficiency can be corrected by enhancing the metabolic capacity for myocardial glucose utilization. This observation provides a basis for further investigation of metabolic modulation as a therapeutic strategy for cardiac diseases leading to heart failure.
Acknowledgments
This work is supported by grants from the National Institute of Health (HL-59246 and HL-67970 to Dr Tian, HL-46033 to Dr Balschi, and DK-45416 and HL-58493 to Dr Kelly). Dr Luptak is a recipient of the postdoctoral fellowship from the American Heart Association Northeastern Affiliate. Dr Tian is an established investigator of the American Heart Association.
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Abstract
Background— Downregulation of peroxisome proliferator–activated receptor- (PPAR) in hypertrophied and failing hearts leads to the reappearance of the fetal metabolic pattern, ie, decreased fatty acid oxidation and increased reliance on carbohydrates. Here, we sought to elucidate the functional significance of this shift in substrate preference.
Methods and Results— We assessed contractile function and substrate utilization using 13C nuclear magnetic resonance spectroscopy and high-energy phosphate metabolism using 31P nuclear magnetic resonance spectroscopy in perfused hearts isolated from genetically modified mice (PPAR–/–) that mimic the metabolic profile in myocardial hypertrophy. We found that the substrate switch from fatty acid to glucose (3-fold down) and lactate (3-fold up) in PPAR–/– hearts was sufficient for sustaining normal energy metabolism and contractile function at baseline but depleted the metabolic reserve for supporting high workload. Decreased ATP synthesis (measured by 31P magnetization transfer) during high workload challenge resulted in progressive depletion of high-energy phosphate content and failure to sustain high contractile performance. Interestingly, the metabolic and functional defects in PPAR–/– hearts could be corrected by overexpressing the insulin-independent glucose transporter GLUT1, which increased the capacity for glucose utilization beyond the intrinsic response to PPAR deficiency.
Conclusions— These findings demonstrate that metabolic remodeling in hearts deficient in PPAR increases the susceptibility to functional deterioration during hemodynamic overload. Moreover, our results suggest that normalization of myocardial energetics by further enhancing myocardial glucose utilization is an effective strategy for preventing the progression of cardiac dysfunction in hearts with impaired PPAR activity such as hearts with pathological hypertrophy.
Key Words: fatty acids glucose metabolism myocardium
Introduction
Peroxisome proliferator–activated receptor (PPAR), a nuclear receptor that regulates the expression of multiple genes controlling both fatty acid uptake and oxidation, is a key player in the transcriptional regulation of substrate preference in the heart.1–7 Downregulation of PPAR, widely observed in diseased hearts such as hearts with cardiac hypertrophy and failure, allows shifting substrate preference from fatty acid to glucose.8 It is not clear whether decreased PPAR activity in hypertrophied hearts is adaptive or maladaptive. Although some studies have suggested that PPAR deficiency renders a fetal metabolic profile and hence impaired functional capacity of the heart, others have shown that reactivation of PPAR worsens contractile function in the hypertrophied and failing heart, suggesting that the downregulation of PPAR is a necessary adaptation.2,3,5,9
Clinical Perspective p 2346
In this study, we sought to determine the functional significance of PPAR deficiency by analyzing cardiac substrate metabolism, myocardial energetics, and contractile function in PPAR–/– hearts. Our findings show that PPAR–/– hearts are able to maintain baseline function but manifest abnormal myocardial energetics, inefficient ATP generation, and impaired contractile function at high workload. These results suggest that a compensatory increase in carbohydrate utilization in response to the downregulation of PPAR occurs at the expense of depleting the metabolic reserve of the heart. Furthermore, by overexpressing a glucose transporter in PPAR–/– hearts, we demonstrate that the inadequate substrate switch can be improved by upregulating the capacity for glucose utilization.
Methods
Animal Models
The PPAR–/– mice (kindly provided by Dr Frank Gonzalez) have been described previously.10 PPAR–/–TG mice were generated by crossing a mouse that overexpresses the insulin-independent glucose transporter GLUT1 (TG) in a cardiac-specific manner11 with PPAR–/– mice to enhance glucose transport in the hearts of PPAR–/– mice. PPAR–/– mice and GLUT1-TG mice (on sv129 and FVB background, respectively) were crossed to generate PPAR+/– and PPAR+/–TG mice. These heterozygous mice were used as breeders. Among the 6 resulting genotypes, PPAR–/–, PPAR–/–TG, TG, and wild type (WT) (1/8 for each genotype expected in every litter) were used for the experiments, and PPAR+/– and PPAR+/–TG (1/4 for each genotype expected in every litter) were used as new breeders.
Isolated Perfused Heart Experiments and 31P Nuclear Magnetic Resonance Spectroscopy
Mice were given heparin (100 U IP) and anesthetized by sodium pentobarbital (150 mg/kg IP). The heart was excised and perfused at a constant pressure of 80 mm Hg at 37°C as previously described.12 The perfusate contains the following (in mmol/L): NaCl 118, NaHCO3 25, KCl 5.3, CaCl2 2, MgSO4 1.2, EDTA 0.5, glucose 5.5, mixed long-chain fatty acids 0.4 bound to 1% albumin, DL--hydroxybutyrate 0.38, lactate 2, and insulin 50 μU/mL equilibrated with 95% O2 and 5% CO2 (pH 7.4). A water-filled balloon was inserted into the left ventricle to record ventricular pressure and heart rate. Balloon volume was adjusted to achieve an end-diastolic pressure of 5 to 10 mm Hg. Isovolumic contractile function was estimated by the product of left ventricular (LV) developed pressure and heart rate (rate-pressure product; RPP). Myocardial oxygen consumption (MVO2) was determined by measuring the coronary flow rate and the PO2 difference between perfusate and effluent from the pulmonary outflow tract.13
After a 30-minute equilibration period, baseline function was recorded, and the perfusion medium was switched to a buffer containing 13C-enriched substrates. To elicit a high contractile performance, concentration of CaCl2 in the perfusate was increased from 2 to 4 mmol/L. Hearts were maintained at either baseline or high workload level for 25 minutes. At the end of the experiments, hearts were freeze clamped with Wollenberger tongs precooled in liquid nitrogen.
31P nuclear magnetic resonance (NMR) spectroscopy of isolated perfused mouse hearts was performed as previously described.12 One 8-minute spectrum was collected at baseline, and 3 consecutive spectra were collected during high workload. In separate hearts, ATP synthesis rates were measured with the 2-site saturation transfer technique by applying a low-power narrow-band radiofrequency pulse to saturate -ATP resonance and observing changes in the Pi resonance area.14,15 Spectra were acquired before (M0)and 4.8 seconds after (M) the saturating pulse was applied by signal averaging 256 total scans as interleaved sets of 8 scans. A complete measurement was acquired in 46 minutes.
13C NMR Spectroscopy
Perchloric acid extracts were prepared from freeze-clamped heart tissue, neutralized by KOH, lyophilized, and subsequently dissolved in 300 μL D2O. Proton-decoupled 13C NMR spectra of tissue extracts were obtained with a Varian Inova 400 spectrometer 3-mm NMR probe. Data were collected using a 60° pulse with a 3.0-second delay, a sweep width of 10 000 Hz, and 16 000 data points. The contributions of each labeled substrate and the total of the unlabeled exogenous and endogenous substrates to the oxidative metabolism were determined using the peak areas of 13C isotopomers at C3 and C4 of glutamate by modeling the tricarboxylic acid (TCA) cycle fluxes as previously described.16,17 To distinguish between utilization of the 5 different substrate groups (glucose, fatty acids, lactate, ketones, and endogenous substrates), hearts were supplied with all substrates, 2 of which were 13C enriched for each perfusion study. In 1 series, [U-13C]fatty acid and [3-13C]lactate were used to determine the relative contributions of fatty acid and lactate. Identical experiments with enriched [U-13C]glucose and [2,4-13C]-hydroxybutyrate determined the relative contribution of these 2 substrates. The contribution from endogenous substrates was determined as the difference from 100%.
High-Performance Liquid Chromatography Measurements and Calibration of 31P NMR Spectra
Freeze-clamped tissues were used to determine the myocardial content of ATP by the high-performance liquid chromatography method as reported previously.18 Myocardial ATP content obtained by high-performance liquid chromatography was converted to [ATP] assuming an intracellular water content of 0.48 mL/g and a protein content of 0.15g/g blotted wet tissue.19 The mean values of [ATP] in hearts freeze clamped at the end of equilibration period were not different among the groups (in mmol/L) (mean±SE; n=4): WT, 11.5±0.8; PPAR–/–, 10.7±0.6; GLUT1-TG, 12.2±1.4; and PPAR–/–TG, 11.9±0.5. These values were used to calibrate ATP peak areas in baseline 31P NMR spectra for the respective groups. The area of the -ATP peak obtained under baseline conditions was set to 100% and used as the reference value for all peaks in all 31P NMR spectra.
Enzyme Activity and Glycogen Measurements
Enzyme activities of phosphofructokinase (PFK),20 GAPDH,21 lactate dehydrogenase (LDH),22 mitochondrial enzyme citrate synthase (CS),23 and cytochrome c oxidase (COX)24 were measured using freeze-clamped heart tissue as previously described. Myocardial glycogen content was determined by measuring the amount of glucose released from glycogen with alkaline extraction used to separate glycogen and exogenous glucose.19 Glucose content in the extract was measured with a Cobas Mira chemistry analyzer (Roche Applied Science). All reagents were purchased from Sigma Chemical Co and were at least analytical grade.
Statistical Analysis
All data are presented as mean±SEM. One-way ANOVA with the Bonferroni post test was performed, and an unpaired t test was used for 2-group comparisons. Analyses were performed with GraphPad Prism version 4.00 for Windows (GraphPad Software). A value of P<0.05 was considered significant.
Results
General Characteristics of the Mice
A total of 173 mice were used for this study; the average age of the animals was 29±1 weeks. Heart weight, body weight, and heart weight normalized to body weight or to tibia length were not different among the groups (Table 1). The volume of the LV balloon was similar when baseline LV end-diastolic pressure was achieved for all hearts (Table 1). Thus, there was no gross cardiac hypertrophy or dilatation in any of the groups.
Substrate Utilization Profile
The relative contributions of carbon fuel substrates to the oxidative metabolism in the heart were measured during baseline and high workload periods (Figure 1). At baseline, the contribution of fatty acids to oxidative metabolism was reduced 3-fold in PPAR–/– hearts compared with WT hearts, and the oxidation of glucose and lactate increased by 3-fold. A similar pattern was seen in PPAR–/–TG hearts at baseline. Increasing GLUT1 expression alone (TG group) led to increased glucose oxidation and decreased fatty acid oxidation, albeit less pronounced than in the PPAR–/– group. The relative contributions from -hydroxybutyrate (ketone) and endogenous substrates (glycogen and triglyceride) were not different.
In WT hearts challenged with high workload, contributions from carbohydrate (glucose and lactate) and ketone increased; the relative oxidation of exogenous fatty acids was unchanged, whereas the contribution from endogenous substrates tended to decrease. In contrast, when PPAR–/– hearts were challenged with high workload, there was no change in the substrate utilization profile compared with baseline, suggesting a loss of metabolic reserve for increasing carbohydrate utilization in these hearts. In PPAR–/–TG hearts, however, glucose oxidation at high workload increased further (up by 50% relative to PPAR–/– at high workload). Thus, increasing glucose transport in PPAR–/– hearts led to a further increase in glucose utilization beyond its intrinsic capacity and partially restored the substrate utilization response to high workload.
Glycogen Concentration and Glycolytic Enzymes
Activities of 3 major glycolytic enzymes, PFK, GAPDH, and LDH, were measured in tissue homogenates of hearts from all groups (Table 2). Despite large differences in glucose oxidation rates, the Vmax of the glycolytic enzymes was not different among the groups. This is consistent with prior observations that parallel changes in flux and enzymatic activity are not obligatory for the glycolytic pathway because the Vmax for glycolytic enzymes far exceeds the flux (>20 times) measured under most circumstances.4,25,26 Indeed, we have previously observed substantially higher glycolytic activity in TG hearts even though the Vmax was unchanged.11
Contractile Performance
We evaluated isovolumic contractile function of all hearts at 2 levels of performance. At the baseline level, all hearts were able to achieve and sustain an RPP of 35 000 mm Hg/min (Figure 3). When switched to a high-performance level, the RPP increased 2-fold in WT hearts and was sustained for the entire 25-minute period. In contrast, PPAR–/– hearts failed to maintain the high contractile performance. At the end of the high-workload period, their RPP was 25% lower than the RPP of the WT hearts (Figure 3). Importantly, contractile function during high workload was normalized in PPAR–/–TG. The contractile function of TG hearts was not different compared with the WT hearts. Decreased fatty acid oxidation in PPAR–/– hearts, despite marked increases in the oxidation of glucose and lactate, was accompanied by the inability to sustain high contractile performance, ie, a loss of contractile reserve. Interestingly, this loss was restored by further enhancing glucose transport and utilization.
Myocardial High-Energy Phosphate Content
To determine whether the altered substrate utilization observed in PPAR–/– hearts impaired high-energy phosphate metabolism leading to contractile dysfunction, we measured high-energy phosphate content and ATP synthesis rate in isolated perfused hearts. The myocardial ATP concentration was not different at baseline for WT and PPAR–/– (11.5±0.8 and 10.7±0.6 mmol/L, respectively; P=NS). During high workload, [ATP] remained fairly constant in the WT group but deteriorated progressively in the PPAR–/– group (Figure 4). The concentration of phosphocreatine (PCr), the energy reserve compound, was lower in the PPAR–/– group at baseline, reflecting a decreased energy reserve. Moreover, despite a reduced contractile performance, [PCr] remained lower during the high workload in PPAR–/– compared with WT hearts. Inorganic phosphate concentration (Pi) increased in all groups at the beginning of the high workload. However, at the end of the protocol, this increase was significantly greater in the PPAR–/– group, reflecting pronounced ATP depletion (Figure 4). The excessive depletion of high-energy phosphate content was corrected in PPAR–/–TG hearts, in which concentrations of both PCr and ATP were restored to the WT level during the high workload (Figure 4). The high-energy phosphate and inorganic phosphate contents of TG hearts were not different compared with the WT hearts. Thus, further enhancing the contribution of glucose to ATP synthesis normalized myocardial energetics in PPAR–/– hearts at high workload.
Oxygen Consumption and ATP Synthesis
The decrease in high-energy phosphate content in PPAR–/– hearts during high contractile performance suggests a defect in the balance of energy supply and consumption. Thus, MVO2 was measured at both levels of workload (Figure 5). MVO2 was similar in all groups at baseline and increased markedly at high workload. Surprisingly, the increase in MVO2 was the greatest in PPAR–/– hearts despite the least increase in contractile performance among the groups. As a result, PPAR–/– hearts had a significantly lower O2 efficiency at high workload (Figure 5). Thus, we measured ATP synthesis rate by an MVO2-independent approach using 31P NMR magnetization transfer technique. Figure 6 shows representative 31P magnetization transfer spectra obtained from a WT heart during high workload. The rate constant for ADP+PiATP was lower in PPAR–/– hearts but was restored in PPAR–/–TG hearts (0.15±0.02, 0.28±0.04, and 0.26±0.02 seconds–1 for PPAR–/–, WT, and PPAR–/–TG hearts, respectively; P<0.05, PPAR–/– versus WT). Using the flux of the reaction, representing the ATP synthesis rate, and MVO2, we calculated the P/O ratio for all groups at high workload (Table 3). Although the P/O ratio in WT is consistent with the reported value in the heart, the ratio was decreased by >50%, suggesting substantial mitochondrial uncoupling in PPAR–/– hearts.27 Oxygen consumption, ATP synthesis, and P/O ratio were not different in PPAR–/–TG and TG groups compared with the WT group. The activities of CS and COX, key enzymes for the TCA cycle and mitochondrial respiratory chain, respectively, were measured. Although CS activity was unaltered, COX activity was increased in the PPAR–/– group (Table 2), suggesting a higher O2 consumption capacity in these hearts.
Taken together, these results suggest that the ATP synthesis rate was lower in the PPAR–/– group at high workload and that MVO2 was paradoxically higher. Intriguingly, the O2 wasting was observed only when the energy demand was substantially raised by driving the heart for maximal performance. Furthermore, these defects could be normalized by increasing glucose utilization, which enabled the PPAR–/–TG hearts to sustain normal myocardial energetics despite decreased fatty acid oxidation.
Discussion
In this study, we showed that the compensatory substrate switch from fatty acids to glucose and lactate in PPAR–/– hearts was not sufficient for maintaining the high-energy phosphate content during high workload. Decreased ATP synthesis during the high workload is likely responsible for the energetic and contractile failure of PPAR–/– hearts. These results suggest that adult hearts with decreased capacity for fatty acid oxidation are more susceptible to progressive deterioration of cardiac function during hemodynamic overload. An important finding of this study is that normalization of high-energy phosphate content by further enhancing glucose utilization is sufficient to rescue these defects.
Decreased PPAR expression and activity contribute to the reappearance of the fetal metabolic pattern, ie, increased reliance on carbohydrates in cardiac hypertrophy and failure.2,3,5,28,29 It has been speculated that this switch in substrate preference may limit the functional capacity of the heart.5 Results from the present study show that hearts lacking PPAR are able to maintain the baseline function, and no gross morphological pathology is found in sedentary mice up to 30 weeks of age. However, myocardial energetics and contractile reserve were reduced in PPAR–/– hearts during high workload challenge, consistent with previous observations that these hearts were unable to cope with stresses.7,30 In this study, we tested the hypothesis that expanding the capacity for glucose utilization is sufficient to compensate for the impaired fatty acid oxidation during high energy demand. Our results support this hypothesis because further increasing glucose transport and utilization has completely rescued the energetic and contractile phenotype of PPAR–/– hearts. Furthermore, our present findings also show that increased reliance on carbohydrates per se is not harmful for the heart. However, a compensatory increase in carbohydrate utilization in PPAR–/– hearts, although sufficient to maintain energy supply for baseline work, exhausted the intrinsic reserve for any further increase in substrate utilization. The inability to increase the contribution of either glucose or fatty acids to oxidative metabolism during high energy demand renders energetic and contractile failure in PPAR–/– hearts subjected to high workload. By overexpressing GLUT1, we were able to enhance glucose utilization and to rescue the heart by 2 mechanisms: (1) increasing glucose uptake, glycolysis, and the contribution of glucose to the TCA flux to a level beyond what can be achieved in nontransgenic hearts and (2) providing more glycogen, a critical substrate for supporting the acute increase in workload.31
31P NMR saturation transfer measurements in this study showed that the ATP synthesis rate of PPAR–/– hearts was 50% lower than WT at high workload. This finding confirms that decreased ATP synthesis is responsible for the progressive loss of ATP and contractile dysfunction of these hearts at high workload. Decreased ATP synthesis at high workload has been observed previously in hypertrophied rat hearts, a model in which PPAR is downregulated.4 Our results show that decreased ATP synthesis can be rescued by further increasing the contribution of glucose to ATP synthesis. These results collectively support the notion that impaired capacity for substrate utilization leads to reduced ATP synthesis in PPAR–/– hearts at high workload. The unexpected finding in the present study is that PPAR–/– hearts waste O2 at high workload. We propose that the oxygen wasting observed here is a consequence of an energy deficit in hearts with impaired capacity for substrate utilization. This hypothesis is based on the following evidence. First, normalizing myocardial energetics by increasing glucose utilization corrected the high MVO2 in PPAR–/– hearts. Second, paradoxically high MVO2 occurs only during high energy demand, unlikely attributable to a constitutive mitochondrial defect. Although our study does not allow further elucidation of the direct causes for oxygen wasting, mitochondrial uncoupling at high workload, as revealed in the present study, will worsen the myocardial energy supply in PPAR–/– hearts and will likely contribute to the eventual contractile failure in PPAR-deficient hearts.
These observations have important clinical implications because downregulation of PPAR has been proposed as a major mechanism for substrate shift in cardiac hypertrophy and failure. Consistent with the notion that substrate switch caused by decreased PPAR activity depletes metabolic reserve, prior studies have shown that cardiac hypertrophy is associated with increased glucose utilization at baseline, but glycolysis fails to increase further during high workload. In addition, overall ATP production from exogenous fuels is decreased.1,4 Here, using transgenic mouse models as a proof-of-concept approach, we demonstrate that metabolic interventions capable of further enhancing glucose utilization have the potential to prevent the vicious cycle of progressive deterioration in pathological cardiac hypertrophy. This concept is also supported by recent studies showing that enhancing glucose utilization by either insulin-dependent or -independent mechanisms improves cardiac function and delays the transition from cardiac hypertrophy to failure.11,32 It needs to be recognized that the genetically modified mice studied here may exhibit other changes in cellular metabolism not defined by the present study. Whether these potential changes contribute to our observations is unknown. Nevertheless, because reactivation of PPAR has been found to cause contractile dysfunction in hypertrophied hearts despite the successful restoration of substrate preference profile, results from this study provide an important alternative strategy to rescue the maladaptive aspect of PPAR deficiency.
In summary, the present study shows that PPAR deficiency reduces the capacity for energy production and cardiac function in response to hemodynamic stress. Furthermore, our findings suggest that the maladaptation to the PPAR deficiency can be corrected by enhancing the metabolic capacity for myocardial glucose utilization. This observation provides a basis for further investigation of metabolic modulation as a therapeutic strategy for cardiac diseases leading to heart failure.
Acknowledgments
This work is supported by grants from the National Institute of Health (HL-59246 and HL-67970 to Dr Tian, HL-46033 to Dr Balschi, and DK-45416 and HL-58493 to Dr Kelly). Dr Luptak is a recipient of the postdoctoral fellowship from the American Heart Association Northeastern Affiliate. Dr Tian is an established investigator of the American Heart Association.
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