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1Department of Nutrition, University of Montreal, Montreal, Quebec; 2Department of Experimental Medicine, McGill University; Montreal, Quebec, Canada; and 3Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio
Submitted 20 July 2004 ; accepted in final form 11 November 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIAL AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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isolated working rat heart perfusion; citric acid cycle; 13C mass isotopomer analysis; fatty acid translocase/CD36; epinephrine
(PPAR), changes that are believed to be a recapitulation of the "fetal gene program" (2, 15). Although reactivation of PPAR in hypertrophied hearts was associated with contractile dysfunction (60), clearly, one potential detrimental consequence of chronic inhibition of FA oxidation, which is currently being intensively investigated, is intracellular lipid accumulation and its associated lipotoxic sequelae (26). Another potential consequence is energy starvation (54). Indeed, despite a slight (11%) increase in the ATP-to-oxygen ratio associated with glucose rather than FA oxidation, it is unclear to which extent energy production from FA oxidation can be fully compensated by enhanced glucose use, especially under conditions of increased energy demand induced by stress. Future investigations appear warranted, given that stress can induce cardiac decompensation in humans with genetic FA oxidation defects (48).
Use of animal models with known alterations in their energy metabolism provides valuable insights on the modulation of the cardiac contractile function by substrate selection. In this regard, the spontaneously hypertensive rat (SHR) is a genetic model of cardiomyopathy associated with hypertension, hypertriglyceridemia, and insulinoresistance (19, 57). The SHR develops cardiac hypertrophy between 9 and 12 wk of age. At 15 wk, the hypertrophy is compensated and hypertension is the unique hemodynamic parameter that differs from control rats (23). Functional symptoms of decompensated cardiomyopathy appear after 18 to 24 mo of age (32). At the metabolic level, SHR hearts show an impaired use of long-chain FA (LCFA) for energy production associated with an enhanced glycolysis (8, 57). Whereas such a metabolic shift appears to suggest a return to the fetal phenotype due to PPAR downregulation (2, 15), this is likely to be an oversimplification of the metabolic status prevailing in the SHR heart. Indeed, some strains of SHR have a genetic defect in the cardiac FA translocase/CD36, a plasma membrane LCFA transporter (19). Interestingly, feeding these SHR strains a diet supplemented with a medium-chain FA (MCFA), a substrate that does not require CD36-facilitated transport, prevented hypertrophy development despite the persistent hypertension (19, 45). Taken together, these data suggest that the reduced exogenous LCFA use in the SHR heart contributes to the development of its cardiomyopathy and is not simply a consequence of the hypertension-induced left ventricular hypertrophy. Nevertheless, under normal ex vivo perfusion conditions, 15-wk-old SHR show cardiac flows, work, and efficiency similar to control Wistar-Kyoto rats (57). However, their capacity to withstand an acute adrenergic stress, a predisposing factor to cardiac decompensation, has not yet been examined.
Hence, a first aim of this study was to test the hypothesis that SHR hearts show an impaired capacity to respond and withstand an acute stress consisting of an adrenergic stimulation compared with control Wistar rat hearts. A second aim was to evaluate the potential role of an impaired LCFA metabolism as a factor determining the heart's resistance to stress. This was achieved by comparing energy substrate selection in SHR and Wistar rat hearts during the stress protocol, and by testing in SHR hearts the effects of MCFA supplementation. We expand on previous studies that characterize the functional and metabolic phenotype of hearts isolated from Wistar, Wistar-Kyoto, and SHR perfused ex vivo in the working mode with 13C-labeled substrates under normal conditions (55, 57). Results from this study demonstrate that exogenous FA oxidation is a factor that determines the resistance of SHR hearts to an acute stress condition.
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MATERIAL AND METHODS |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The sources of chemicals, biological products, and 13C-labeled substrates as well as the procedure for the dialysis of BSA FA-free (BSA fraction V; Intergen) have been described previously (11, 12, 55–57). The stock solution of 10 mM epinephrine [bitartrate salt; Sigma-Aldrich (St. Louis, MO), in nitrogen gassed water] was prepared fresh before use.
Heart Perfusions in Semirecirculating Working Mode
Animal experiments were approved by the local animal care committee in compliance with the guidelines of the Canadian Council on Animal Care. Male SHR (15 wk old) and Wistar rats (300–330 g; Charles River) were provided with food and water ad libitum. The procedure for heart isolation and its ex vivo perfusion in the working mode have been described previously in detail (55, 57). The only modification that was made concerned the aortic afterload pressure, which was set at 80 mmHg for Wistar rat hearts and either 80, 95, or 110 mmHg for SHR hearts. Atrial preload was maintained at 11.5 mmHg. As in previous studies, the following functional parameters were continuously monitored throughout the entire perfusion period: 1) atrial inflow and the aortic outflow, with calibrated electromagnetic flow probes (model FM501; Carolina Medical Electronics); 2) temperature, with a thermocouple (Yellow Springs Instruments); and 3) left ventricular contractile functions, namely heart rate, maximum left ventricular pressure, left ventricular end-diastolic pressure, and maximum value for first derivative of left ventricular pressure (+dP/dtmax), with a pressure transducer connected to a polyethylene catheter, pulled through the ventricular wall and anchored at the apex of the heart by a fluted end (Digi-Med Heart Performance Analyzer; Micro-Med).
Perfusion Protocols
Two different perfusion protocols were followed to 1) compare the functional and metabolic responses of SHR and control Wistar rat hearts to an acute epinephrine stimulation, and 2) evaluate the effects of MCFA supplementation in SHR hearts under the stress condition.
Protocol 1. Hearts from SHR and Wistar rats were perfused with a semirecirculating modified Krebs-Henseleit bicarbonate buffer containing physiological concentrations of substrates (in mM: 5.5 glucose, 1 lactate, 0.2 pyruvate, 0.4 oleate bound to 3% albumin, and 0.1 EDTA, plus 4 nM insulin, 50 µM carnitine, and 5 nM epinephrine). The ionized calcium concentration of the albumin-containing buffer was determined to be 1.8 mM. Lactate and pyruvate were added in a physiological ratio to minimize perturbations of the cytosolic redox state that occur when lactate is supplied alone. Addition of carnitine compensates for its potential loss during heart isolation (43). We have previously shown that hearts from SHR and Wistar rat hearts perfused under basal conditions with the aforementioned buffer maintain stable cardiac functions (55, 57). The 40-min perfusion protocol consisted of a 10-min basal prestress period (T0-T10) followed by a 30-min stress period during which the epinephrine concentration in the perfusion buffer was raised to 10 µM (T10-T40). The aortic afterload pressure was 80 mmHg for Wistar rat hearts (n = 8) and either 80 mmHg (SHR80, n = 5), 95 mmHg (SHR95, n = 9), or 110 mmHg (SHR110, n = 4) for SHR hearts. The higher afterloads for SHR heart perfusions were chosen to reproduce the higher peripheral resistance due to hypertension.
Protocol 2.
SHR hearts were perfused at an afterload pressure of 95 mmHg as in protocol 1, except that the FA concentration in the perfusion buffer was raised by the addition of either 0.09 mM oleate (LCFA-group, 0.49 mM oleate, n = 9) or 0.2 mM octanoate (MCFA-group, 0.4 mM oleate + 0.2 mM octanoate, n = 9). The total concentration of exogenous FA was calculated to provide the same quantity of acetyl-CoA units, assuming complete 
-oxidation.
Biochemical and Metabolic Parameters
For any given perfusion, one or more unlabeled substrates were replaced by their corresponding 13C-labeled substrates at the same concentration, either [U-13C3]lactate and [U-13C3]pyruvate (n = 4–5 per group), [1-13C]oleate (n = 4 per group), or [1-13C]octanoate (n = 4). All 13C-labeled substrates were supplied at 99% enrichment. Throughout the perfusion, influent and effluent perfusates were collected 1) every 5 min to assess lactate dehydrogenase (LDH) release and 2) at T7-T10 (prestress) and T37-T40 (stress) to assess citrate and succinate release rates as well as lactate and pyruvate uptake and production rates. At the end of the perfusion, the heart was freeze-clamped with metal tongs chilled in liquid nitrogen, weighed, and stored at –80°C for subsequent analyses.
Analytical Procedures
The 13C enrichment and concentrations of the citric acid cycle (CAC) intermediates (citrate, isocitrate, -ketoglutarate, succinate, fumarate, and malate), activities of selected CAC (citrate synthase, aconitase, and NAD-isocitrate dehydrogenase), and -oxidation [carnitine palmitoyl transferase-1 (CPT-1), and medium-chain acyl-CoA dehydrogenase (MCAD)] enzymes as well as flux ratios relevant to substrate selection for citrate synthesis namely pyruvate and FA, from 13C enrichment of the acetyl [carbon 4 and 5 (C4+5)] and oxaloacetate (OAA; C1+2+3+6) moiety of citrate (OAACIT), were measured in powdered tissue samples. Conditions and procedures for the operation and analysis of all metabolites by gas chromatography-mass spectrometry (GC-MS) were previously described (11, 12, 55, 57). Briefly, succinate and citrate release rates were quantified by isotope dilution GC-MS and flow measurements (11, 12, 55–57). Concentrations and 13C-labeled mass isotopomer distribution (MID) of CAC intermediates and related metabolites (citrate and its OAA moiety, -ketoglutarate, succinate, fumarate, malate, and pyruvate), as well as the 13C-labeled MIDs of lactate and pyruvate in influent and coronary effluent perfusates treated with 1 M NaB2H4, were determined by GC-MS assays (11, 12, 55–57). Perfusate lactate concentrations, LDH release, tissue citrate synthase, aconitase, and NAD-isocitrate dehydrogenase activities were determined by enzymatic assays with a Roche Cobas Fara spectrophotometer as detailed in our previous studies (11, 12, 55–57). The maximal enzyme activities of CPT-1 and MCAD were measured in powdered tissue samples by radioactive method as previously described (22, 37). Protein contents were measured with a Bio-Rad kit with BSA serving as a standard. Citrate synthase activity was expressed as units per gram of total protein, where 1 unit of enzyme activity is defined as the amount necessary to catalyze the conversion of 1 µmol substrate/min at 37°C. The other enzyme activities were expressed relative to the citrate synthase activity.
Calculations
Myocardial oxygen consumption, intracellular pH, and functional parameters.
Free Ca2+, PO2, and PCO2 were determined in influent and coronary effluent perfusate samples collected on ice at 8 and 38 min with a blood gas, electrolytes, and pH analyzer (ABL 77 series; Radiometer). Myocardial oxygen consumption (M
O2; µmol/min), intracellular pH (pHi), the rate-pressure product (RPP; mmHg·beats·min–1), the cardiac power (mW), and the cardiac efficiency (mW·µmol O2–1·min–1) were calculated from previously reported equations (57).
Flux parameters.
GC-MS data are expressed as molar percent enrichment (MPE) as defined previously (11, 12, 55–57). Briefly, mass isotopomers of metabolites containing 1 to n 13C-labeled atoms were identified as Mi with i = 1, 2,... n, and the absolute MPE of individual 13C-labeled mass isotopomers (Mi) of a given metabolite was calculated as MPE (Mi) = % AMi/[AM + 
AMi], where AM and AMi represent the peak areas from ion chromatograms corrected for natural abundance, corresponding to unlabeled (M) and 13C-labeled (Mi) mass isotopomers, respectively. The equations to calculate flux ratios relevant to citrate synthesis (CS) in hearts perfused with [U-13C3]lactate and [U-13C3]pyruvate, [1-13C]oleate, or [1-13C]octanoate have been described previously in detail (11, 12, 55–57). In brief, flux ratios were calculated from the measured MID of the following tissue metabolites: 1) OAACIT, from which we extrapolated the acetyl moiety of citrate (ACCIT), 2) pyruvate and, 3) succinate. In this study, we reported the following flux rates, expressed relative to that of CS: 1) oleate oxidation: OLE/CS = 9 x M1 ACCIT/M1 oleate (Eq. 3 of Ref. 56), 2) octanoate oxidation: OCT/CS = 4 x M1 ACCIT/M1 octanoate (Eq. 6 of Ref. 11), 3) pyruvate decarboxylation (PDC): PDC/CS = M2 ACCIT/M3 pyruvate (Eq. 5 of Ref. 11), 4) pyruvate carboxylation (PC): PC/CS = M3 OAACIT/M3 pyruvate (Eq. 4 of Ref. 11), and 5) the contribution of other substrates (OS), such as endogenous FA and/or amino acids, to the formation of acetyl-CoA: OS/CS = 1 – (PDC/CS + OLE/CS + OCT/CS). Measured MPE M3 OAACIT was corrected for the fraction of M3 OAA molecules coming from citrate isotopomers metabolized in the CAC as described in Eqs. 8–10 of Ref. 11. To extrapolate the MPE M1 and M2 of ACCIT from the measured MID of citrate and its OAA moiety, we used Eqs. 18–19 of Ref. 11.
Lactate and pyruvate uptake and production rates. Rates of production and uptake of lactate and pyruvate (µmol·min–1·gww–1) were obtained by multiplying their perfusate concentration of unlabeled (M, M1) and 13C-labeled (M3, M4) (µmol/ml) isotopomers, respectively, by the coronary flow (ml/min), as previously described (55, 57). Values are reported to the heart's wet weight in grams (gww).
Absolute CAC flux rate.
The absolute CAC flux rate (nmol·min–1·gww–1) was calculated from the previously reported equation: CAC flux = MO2 x [(PDC/CS x 0.333) + (OLE/CS x 0.353) + (OCT/CS x 0.364) + (OS/CS x 0.351)] (Eq. 3 in Ref. 55). This equation is based on values of 1) MO2 (nmol/min) and 2) substrate flux ratios, which are multiplied by a factor that corresponds to the amount of citrate formed (nmol) per oxygen consumed, namely, 0.333 for carbohydrates, 0.353 for exogenous oleate, 0.364 for octanoate, and 0.351 for the other substrates, which we assumed to be endogenous triglyceride (TG) stores consisting of equal proportion of oleate and palmitate. Values were expressed in terms of grams wet weight.
Statistical Analysis
Data are expressed as means ± SE of n = 4–9 heart perfusions and are compared with those obtained for the control group, i.e., Wistar rat group (protocol 1) and LCFA-group (protocol 2). Student's unpaired t-test was applied to compare the body and cardiac weight (Wistar vs. compiled data of the SHR groups) and metabolic data (protocol 1, Wistar vs. SHR95; protocol 2, LCFA- vs. MCFA-group). One-way (prestress) or two-way (stress values) ANOVA followed by a Bonferroni selected-comparison test in the protocol 1 was applied for statistical evaluation of functional and biochemical parameters. A probability of P < 0.05 was considered to be significant.
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RESULTS |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Mean body weights were similar for 15-wk-old SHR and Wistar rats (331 ± 6 and 318 ± 8 g, respectively, P = 0.25). However, the SHR had a higher cardiac wet weight (2.01 ± 0.07 vs. 1.68 ± 0.06 g, P < 0.01) and, consequently, an increased heart-to-body weight ratio (6.07 ± 0.14 vs. 5.31 ± 0.19 mg/g, P < 0.01), an index of cardiac hypertrophy (8, 19, 34, 58). There was no significant difference in body weight or cardiac wet weight among the various SHR groups in protocols 1 or 2 (data not shown).
Effect of an Acute Adrenergic Stress in Working SHR vs. Wistar Rat Hearts
Functional and physiological parameters. Values for selected cardiac functional and physiological parameters documented during the prestress and stress periods are reported in Table 1, Figs. 1 and 2. Because Wistar rat and SHR showed differences in heart weight with a similar body weight, values for metabolically relevant parameters shown in Table 1 are expressed both per heart rate and per gram wet weight.
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O2 (Fig. 1G and Table 1), which is consistent with the workload-induced increase in energy demand. It is noteworthy that SHR hearts perfused at 80 or 110 mmHg showed values of coronary flow and MO2 that differed significantly from that of Wistar rat hearts, but the difference remained significant only for the coronary flow of the SHR80 group when values were expressed per gram wet weight (Wistar 12.7 ± 0.7 vs. SHR80 8.4 ± 0.5, P < 0.01; SHR95 10.6 ± 0.6, not significant; and SHR110 14.8 ± 1.3 ml·min–1·gww–1, not significant, respectively). Stress period. Addition of 10 µM epinephrine markedly and differentially affected most functional parameters measured in both Wistar rat and SHR hearts. Because several parameters differed between perfusion groups in the prestress period, data for selected functionally relevant parameters [left ventricular developed pressure (LVDP), +dP/dtmax, and cardiac power]) are also expressed as a percentage of their prestress values to clearly delineate the effects of stress (Fig. 2).
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With respect to cardiac flow parameters, addition of 10 µM epinephrine elicited a time-dependent decline in the aortic flow (Fig. 1E) and cardiac output (data not shown) in both groups, which was significantly more drastic in SHR hearts and independent of the afterload pressure. At the end of the stress period, aortic flow values were almost 1.5-fold lower in SHR than in Wistar rat hearts. Cardiac power (Figs. 1F and 2C), which reflects both cardiac pressures and flows, was also more severely depressed in SHR hearts, at all afterload pressures, compared with control Wistar rat hearts.
Epinephrine addition did not affect the coronary flow or MO2 in any of the four groups despite the increased workload (Fig. 1G and Table 1). Nevertheless, calculated values of pHi, at the end of the stress period, varied between 7.33 and 7.40 and did not suggest tissue ischemia. Finally, cardiac efficiency, which was initially similar in all groups, was decreased at the end of the stress period by 25 ± 8% in Wistar rat hearts and by 34 ± 3, 42 ± 3, and 38 ± 3% in the SHR80, SHR95, and SHR110 hearts, respectively (P = 0.09).
As depicted in Fig. 1H, the release rates of LDH, an index of tissue damage (40), were similar during the prestress period in all four groups. Addition of 10 µM epinephrine induced a progressive increase in the LDH release, which reached a plateau at 30 min for Wistar rat hearts but continued to rise in SHR hearts at all afterload pressures.
Taken altogether, the aforementioned data demonstrate that SHR hearts have an impaired functional response to stress at all afterload pressures. They also highlight the greater susceptibility of SHR hearts to the epinephrine-induced acute heart failure, which was accompanied by more extensive tissue damage.
Metabolic and Flux Parameters
SHR hearts perfused at an afterload of 95 mmHg were selected for subsequent characterization of the metabolic response of SHR hearts to a stress challenge and of the effects of MCFA. Compared with the SHR110 perfusion group, the SHR95 group showed values similar to the control Wistar rat heart for a greater number of cardiac functional parameters. All metabolic data were expressed relative to cardiac wet weight to take into account the higher cardiac weight of SHR hearts.
As shown in Fig. 3, lactate and pyruvate uptake rates were similar for SHR95 and Wistar rat hearts and were not affected by the adrenergic stimulation. Although lactate and pyruvate were supplied at the physiological ratio of 5:1, their uptake rates were almost similar, indicating a preferential pyruvate uptake. Lactate production rate was slightly, although not significantly, higher in SHR95 hearts during the prestress period. Addition of 10 µM epinephrine raised lactate production rates in both groups but to a greater extent in the SHR95 group, suggesting a higher glycolytic activity. The lactate-to-pyruvate production ratio was similar in the prestress period but became significantly higher in SHR95 hearts at the end of the stress period (11.4 ± 1.2 vs. 5.9 ± 0.5, P < 0.05), suggesting a more reduced cytosolic state.
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50% from exogenous lactate and pyruvate, was similar between the SHR95 and the Wistar group (Fig. 4), whereas the contribution of exogenous oleate was depressed more than twofold in SHR95 hearts (16 ± 1 vs. 44 ± 4%, P < 0.001). Consequently, the contribution of other endogenous substrates was increased, although it remained negligible in control Wistar hearts (20 ± 4 vs. 1 ± 4%, P < 0.01). The absolute CAC flux rate, calculated from MO2 and flux ratios, was significantly lower in SHR95 compared with Wistar rats (2,185 ± 151 vs. 2,785 ± 172 nmol·min–1·gww–1, respectively, P < 0.05).
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-ketoglutarate, succinate, fumarate, and malate) or their total levels (SHR95, 823 ± 41 vs. Wistar, 798 ± 59 nmol/gww, P = 0.73). Similarly, there were no difference in the maximal activities of the mitochondrial CAC enzymes, citrate synthase (SHR95, 3.33 ± 0.05 vs. Wistar, 3.14 ± 0.20 U/mg protein, P = 0.30), aconitase, and NAD+-isocitrate dehydrogenase, assessed in homogenized hearts freeze-clamped at the end of the stress period (Fig. 6). As for the -oxidation enzymes CPT-1 and MCAD, the activity (expressed relative to CS) was increased in SHR compared with Wistar rat hearts for MCAD (0.1058 ± 0.0016 vs. 0.0830 ± 0.0018, P < 0.0001), but not for CPT-1 (13.4 ± 1.8 vs. 10.8 ± 1.2, P = 0.24).
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Effects of MCFA in Working SHR Hearts Subjected to an Acute Adrenergic Stress
Functional and physiological parameters. In a second set of experiments (protocol 2), SHR hearts were perfused at an aortic afterload pressure of 95 mmHg and subjected to the same stress protocol, but the FA concentration in the perfusion buffer was raised by the addition of either 0.09 mM oleate (LCFA-group) or 0.2 mM octanoate (MCFA-group). Assuming complete oxidation, these two concentrations of FA would generate equivalent amounts of acetyl-CoA units.
Values for the various functional and physiological parameters measured for the LCFA-group hearts were similar to those shown for the SHR95 group in Table 1, and Figs. 1 and 2 (data not shown). Addition of 0.2 mM octanoate improved the LVDP (Fig. 7A) and reduced the total LDH release 1,890 ± 89 to 1,145 ± 155 mU/min (P < 0.001) (Fig. 7B). However, all other measured functional and physiological parameters were similar to those reported in Table 1 and Figs. 1 and 2 (data not shown).
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Second, the relative flux through anaplerotic pyruvate carboxylation (PC/CS flux ratio) was 2.3-fold greater in the MCFA-group (0.116 ± 0.027 vs. 0.049 ± 0.006, P = 0.05). In agreement with this enhanced anaplerosis, without changes in the rate of efflux of citrate and succinate, total tissue level of CAC intermediates, and specifically that of isocitrate and malate, was increased in the MCFA- compared with the LCFA-group (Fig. 9).
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DISCUSSION |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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downregulation (2, 15), clearly, this is an oversimplification of the metabolic situation in SHR hypertrophied hearts. Indeed, SHR also have a genetic deficiency in the FA translocase/CD36, which according to the recent study by Hajri et al. (19), is a primary determinant of hypertrophy. Results from the present study demonstrate that the contribution of exogenous FA oxidation to energy production is a factor determining the functional and metabolic responses of working SHR hearts, perfused ex vivo with a mixture of substrates mimicking the in situ milieu, to an acute stress.
As our model of acute stress, we chose adrenergic stimulation with 10 µM epinephrine. This concentration was selected to induce a maximal adrenergic stimulation in SHR hearts (34), taking into account the potential reduction in the number of -adrenergic receptors as well as desensitization of the -adrenergic response in SHR hearts [for review, see Castellano and Bohm (6)]. This acute adrenergic stimulation induced the expected chronotropic and inotropic effects and, consequently, increased the cardiac work in control Wistar hearts, thereby concurring with data of others (9, 17, 18). However, compared with control Wistar rat hearts, working SHR hearts showed an impaired capacity to functionally respond and to withstand a 30-min infusion of 10 µM epinephrine. The impaired response of SHR hearts was evidenced by the lack of increase in the developed pressures and a smaller rise in the values of +dP/dtmax at all afterload pressures. SHR hearts also showed an impaired capacity to withstand the acute adrenergic stimulation, as reflected by a more rapid and severe decline in the aortic flow, associated with an enhanced LDH release. It is noteworthy that hypertrophied SHR hearts exhibit distinct features compared with models of experimentally induced pressure overload hypertrophy in that they show little or no change in myosin heavy-chain isoform distribution and expression/activity of calcium handling proteins (45, 50). However, alterations were reported in protein kinase A-dependent troponin phosphorylation, which could contribute to the impaired response to sympathetic stimulation (30).
Our functional data in working SHR hearts subjected to acute adrenergic stimulation differ to some extent from that of Noresson et al. (34) who reported a 75% increase in +dP/dtmax after a 3 µM epinephrine stimulation similar to control hearts. This discrepancy could be explained by the substrate composition of the perfusion buffer, namely glucose alone in the study by Noresson et al. (34) versus glucose, lactate, pyruvate, and LCFA/albumin to better mimic the in situ milieu in our study. Substrate supply is known to modulate cardiac functions and energetics under various conditions (5). Another difference between these two studies is the epinephrine concentration, 3 vs. 10 µM. However, the functional response of the normal heart was reported to be unaffected by epinephrine concentration in the 1 to 10 µM range (9), in contrast to LDH release (20).
At the metabolic level, working SHR hearts subjected to acute adrenergic stress showed, compared with Wistar rat hearts, the following alterations, which were similar to those reported previously in the absence of stress (57): 1) a reduced contribution of exogenous LCFA to energy production, which was not compensated by carbohydrates but by endogenous sources, either LCFA released from TG stores or amino acid from proteolysis; and 2) enhanced lactate and citrate release rates. Although the various results of this study do not provide a definite explanation for the differential capacity of the SHR and Wistar rat hearts to respond and withstand an acute adrenergic stress, they nevertheless highlight some potentially contributing metabolic factors. These factors include an ischemic component, alterations in cytosolic redox regulation, and LCFA use, and will be sequentially discussed below. The emphasis of the discussion will be on the latter factor because it was modulated by MCFA addition.
Our data suggest that in our stress model, the ischemic component was small and similar in both SHR and Wistar rat hearts, although hypertrophied SHR hearts could be more susceptible to ischemic injury (16). Indeed, changes in calculated pHi and succinate release were marginal compared with those reported previously using a low-flow ischemia protocol mimicking hibernation (10). A lower coronary flow reserve in working SHR hearts due to a dysfunctional constitutive nitric oxide synthase cannot be excluded (59), although coronary flow and MO2 were augmented linearly (4) in response to a raise in the afterload pressure from 80 to 110 mmHg (Fig. 1G). It is noteworthy that the increase in lactate release and in the lactate-to-pyruvate ratio in SHR hearts at the end of the stress period, do not necessarily reflect myocardial ischemia (35). Indeed, O'Donnell et al. (35) presented evidence for a limitation in the transfer of cytosolic NADH to mitochondria when glycolytic flux is enhanced in normal hearts subjected to high workload. This limitation could become even more important in SHR hearts, which have an intrinsically elevated glycolytic activity associated with increased mRNA levels for phosphofructokinase and LDH (8, 21, 57). Nevertheless, the increased cytosolic [NADH]/[NAD+] ratio could potentially impact the SHR heart's capacity to respond and withstand an adrenergic stress. Indeed, this has been associated with a decreased cytosolic phosphorylation potential, i.e., [ATP]/[ADP]·[Pi] ratio (25), and a reduced calcium transport in the sarcoplasmic reticulum (29), possibly through an inhibitory effect of cytosolic NADH on cardiac ryanodine receptors (61). Furthermore, it may favor free radical production by 1) activating cardiomyocyte NAD(P)H oxidase (31), an enzyme that is more highly expressed in SHR hearts (41); or 2) inducing the formation of the unstable ubisemiquinone radical (49).
Regarding the impaired lipid metabolism of SHR hearts, our 13C-labeling data show a more than twofold decrease in the contribution of exogenous oleate oxidation to CS, whereas the contribution of carbohydrates of all sources, which include glycogen, is unchanged. Consequently, in SHR hearts, the contribution of other, unaccounted, endogenous sources reaches values as high as 20%, whereas it is negligible in control Wistar hearts. A downregulation of the expression of FA oxidation enzyme genes, especially the MCAD gene, together with enhanced glycolysis, is a metabolic pattern observed in several other animal models of cardiomyopathy, as volume- or pressure-overloaded rodents (2, 47), or in human hypertensive cardiomyopathy (13), consistent with the reinduction of a fetal phenotype (2). However, the higher MCAD activity and normal CPT-1 activity, as well as higher levels of CPT-1 and CPT-2 mRNA, documented in 15-wk-old hypertrophied SHR hearts (Fig. 6 and Ref. 21, respectively) suggest the presence of other, yet-to-be identified, regulatory mechanism(s), which could compensate CD36 deficiency. One potential mechanism is activation of AMP-dependent protein kinase due to high circulating levels of leptin or angiotensin II in SHR (7, 33, 41, 53).
Nevertheless, together our results support the hypothesis of an inherited defect in the FA translocase/CD36 (19) as the main determinant for the reduced LCFA use in SHR hearts. The potential detrimental consequences of such a lower exogenous LCFA use for the SHR hearts was emphasized by recent studies, which demonstrated that feeding SHR with an MCFA-supplemented diet, a substrate that does not require CD36-facilitated transport, prevented hypertrophy development despite persistent hypertension (19, 45). In the present study, the replacement of 0.09 mM oleate (total concentration 0.49 mM) by 0.2 mM octanoate (MCFA-group, protocol 2) reduced the cardiac tissue damage of working SHR hearts subjected to acute adrenergic stimulation, as reflected by a 40% decrease in LDH release rate, and improved, although only slightly, the cardiac mechanical functions.
The major metabolic impact of MCFA addition to SHR hearts subjected to stress appears to be a modulation of energy substrate selection and anaplerosis rather than a change in the rate of acetyl-CoA formation for CS. Indeed, the contribution of exogenous FA was increased twofold, principally due to a direct contribution of MCFA (10% of total CAC flux), whereas that of exogenous oleate remained unchanged. The increased contribution of exogenous FA was associated with an equivalent decrease in the contribution of other, unaccounted for, endogenous sources. There were no changes in the contribution of carbohydrates, which include all exogenous and endogenous sources (i.e., glucose, lactate, pyruvate, and glycogen). There was also no change in the absolute CAC flux rates calculated by using (Eq. 3 in Ref. 55), which suggests unchanged total ATP production from mitochondrial substrate oxidation. However, as discussed below, these calculations are subject to some uncertainties.
Further work is necessary to elucidate the nature of these endogenous substrates, but TG stores appear as more plausible candidates than proteins. The reported contribution of TG to total ATP production in the heart appears to vary between 15 and 50% in isolated working rat hearts perfused in the presence and absence of exogenous FA, respectively (26, 46). Epinephrine was shown to induce maximal use of endogenous TG and limited mono- and diglyceride (DG) recycling (52), an effect that depended also on the exogenous FA concentrations. In fact, cycling among the various intracellular lipid pools appears to be regulated in a complex fashion to adjust mobilization of endogenous FA in accord with their exogenous availability (26, 52). SHR hearts appear to have a defect in the conversion of DG to TG, attributed to their lack of CD36 transporter (19). A dysregulation in intracellular lipid cycling could activate futile cycles that increase proton production and energy demand of myocardial metabolism by 30% (24, 28). This could also lead to the accumulation of palmitate released from endogenous TG stores, which was shown to induce cardiac cell death when it is oxidized, although octanoate and oleate did not (14). Finally, this could change the concentration of diacylglycerol which is a lipid second messenger capable of activating intracellular signaling pathways via a stimulation of protein kinase C (38). Their increased intracellular concentration enhances cardiomyocyte contractility (38) and resistance to ischemia with a reduction in cardiac necrosis (1). Interestingly, hexanoate, a six-carbon FA, has been shown to improve reperfusion recovery in isolated perfused rat hearts via a mechanism involving inhibition of intracellular TG lipolysis (28). Hence, whether a reduced contribution of TG-derived LCFA to energy metabolism with octanoate is part of the mechanism that contributes to a reduction in cardiac tissue damage in stressed SHR hearts appears to be a possibility that is worth investigating in future studies.
Addition of octanoate to working SHR hearts subjected to stress also induced an increase in tissue levels of total CAC intermediates and, specifically, of isocitrate and malate. Such an effect has been previously observed in unstressed normal hearts (10, 36, 39, 55). In our study, the citrate and succinate release rates were not modified in the presence of octanoate, suggesting that mitochondrial CAC efflux was not decreased. The increase in PC/CS flux, which reflects anaplerosis via pyruvate carboxylase and/or malic enzyme, is consistent with the raise in tissue malate. A preferential shuttling of pyruvate to carboxylation rather than decarboxylation, as reflected by the PC/PDC flux ratio, has been observed by others in normal hearts (36, 55). Other anaplerotic pathways have not been investigated in this study, but their contribution, if any, should be minor in the absence of dilution in the 13C enrichments of CAC intermediates by the entry of unlabeled carbons (dilution factor, calculated from Eq. 10, Ref. 11; Wistar 1.05 ± 0.02 vs. SHR 1.03 ± 0.04, P = 0.66). Whatever the anaplerotic source, the increase in CAC catalytic units could contribute to improve cardiac energy metabolism in presence of a defective LCFA use, as it occurs in human genetic FA oxidation defects (44) by a yet-to-be identified mechanism.
We recognize that several considerations should be kept in mind in this study. First, there is a possibility that a greater beneficial effect of MCFA on the cardiac function and tissue damage could have been observed at a lower dose of epinephrine and/or a higher dose of octanoate. Second, regarding our flux measurements, although the decline in aortic flow suggests a non-steady-state condition, we believe that our 13C-labeling method provides reliable values for the following reasons. First, coronary flow and citrate release rates were constant, and many functional (HR, LVDP) parameters were only marginally affected throughout the perfusion protocols. Second, we previously determined in other heart perfusion experiments that 13C enrichments of the acetyl-moiety of citrate reached plateau values in 5 min (Vincent G and Des Rosiers C, unpublished observations). Third, the reported absolute CAC flux rates, which are calculated from MO2 values, could be overestimated, especially in SHR hearts. In fact, we cannot evaluate the percentage of oxygen leak associated with free radical production, a process that is enhanced in SHR hearts (3). Finally, it is noteworthy that the extrapolation of an ATP yield from the calculated CAC flux rates assumes that FA oxidation is complete and occurs predominantly in mitochondria rather than in peroxisomes (42). The validity of this assumption under our conditions is supported by our finding in two additional perfusions with SHR hearts using [U-13C18]oleate that the contribution of exogenous LCFA was similar to that assessed by using [1-13C]oleate (data not shown).
In conclusion, the results of this study demonstrate that the 15-wk-old SHR heart has an impaired capacity to withstand an acute adrenergic stress, which can be improved by increasing the contribution of exogenous FA oxidation to energy production by MCFA supplementation. Whereas a restricted capacity for energy production from exogenous LCFA does not affect the SHR heart's function under basal condition (57), it appears to impair the heart's capacity to withstand a stress challenge, which increased the energy demand. Within the context of the development of new drugs modulating the heart's substrate preference, our data raise the question as to whether a critical threshold of exogenous FA use should be preserved for optimal energy production through oxidation and/or cycling of intracellular lipid stores. In this regard, administration of short and/or medium chain FA could be beneficial, especially when insulin resistance reflects defects in FA uptake and use.
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ACKNOWLEDGMENTS |
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This work was presented at the Heart Failure 2003 Congress held in Strasbourg, France, June 21–24, 2003, and at the Society for Heart and Vascular Metabolism held in Freiburg, Germany, June 18–20, 2003.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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