Identification of subcellular energy fluxes by P NMR spectroscopy in the perfused heart: contractility induced modifications of energy transfer pathways

The identification of subcellular fluxes of exchange of ATP, phosphocreatine (PCr) and Pi between mitochondria, cytosol and ATPases and pathways of energy transfer in a whole organ is a challenge specially in the myocardium where 50% of creatine kinases (CK) are found in close vicinity of ATP producing (mito-CK) and utilizing ( MM-bound CK) reactions. To dissect their contribution in cardiac energy transfer we recently developed a new experimental³¹P NMR spectroscopy approach. This led to identify three kinetically different subcellular CKs and to evidence experimentally the CK shuttle in a rat heart perfused in isovolumy. Here we show that a decreased energy demand alters energetic pathways : two CKs (cytosolic and MM-bound) functioning at equilibrium and a non mitochondrial ATPPi exchange was sufficient to describe NMR data. Mito-CK fluxes was not detected anymore. This confirms the dependence of energy pathways upon cardiac activity. Indeed the subcellular localization and activity of CKs may have important bioenergetic consequences for the in vivo control of respiration at high work: free ADP estimated from global CK equilibrium might not always adequately reflect its concentration at the ANT.     

Carbon flux through citric acid cycle pathways in perfused heart by 13C NMR spectroscopy

Mathematical models of the TCA cycle derived previously for 14C tracer studies have been extended to 13C NMR to measure the 13C fractional enrichment of [2-13C]acetyl-CoA entering the cycle and the relative activities of the oxidative versus anaplerotic pathways. The analysis is based upon the steady-state enrichment of 13C into the glutamate carbons. Hearts perfused with [2-13C]acetate show low but significant activity of the anaplerotic pathways. Activation of two different anaplerotic pathways is demonstrated by addition of unlabeled propionate or pyruvate to hearts perfused with [2-13C]acetate. In each case, the amount of [2-13C]acetate being oxidized and the relative carbon flux through anaplerotic versus oxidative pathways are evaluated.     

31P NMR detection of subcellular creatine kinase fluxes in the perfused rat heart: contractility modifies energy transfer pathways

The subcellular fluxes of exchange of ATP and phosphocreatine (PCr) between mitochondria, cytosol, and ATPases were assessed by (31)P NMR spectroscopy to investigate the pathways of energy transfer in a steady state beating heart. Using a combined analysis of four protocols of inversion magnetization transfer associated with biochemical data, three different creatine kinase (CK) activities were resolved in the rat heart perfused in isovolumic control conditions: (i) a cytosolic CK functioning at equilibrium (forward, F(f) = PCr --> ATP, and reverse flux, F(r) = ATP --> PCr = 3.3 mm.s(-1)), (ii) a CK localized in the vicinity of ATPases (MM-CK bound isoform) favoring ATP synthesis (F(f) = 1.7 x F(r)), and (iii) a mitochondrial CK displaced toward PCr synthesis (F(f) = 0.3 and F(r) = 2.6 mm.s(-1)). This study thus provides the first experimental evidence that the energy is carried from mitochondria to ATPases by PCr (i.e. CK shuttle) in the whole heart. In contrast, a single CK functioning at equilibrium was sufficient to describe the data when ATP synthesis was partly inhibited by cyanide (0.15 mm). In this case, ATP was directly transferred from mitochondria to cytosol suggesting that cardiac activity modified energy transfer pathways. Bioenergetic implications of the localization and activity of enzymes within myocardial cells are discussed.     

The steady-state rate of ATP synthesis in the perfused rat heart measured by 31P NMR saturation transfer

The steady-state rate of ATP synthesis in the isolated, Langendorff-perfused rat heart was determined using a ³¹P NMR saturation transfer method. At 37°C and a perfusion pressure of 70 cm H₂O the value is 2.8 ± 0.3 (n=5 ± S.E.M.) μmol.s^?1. (g. dry wt.)^?1. The activity of creatine phosphokinase measured in the same experiments was 14.6 ± 1.0 μ mol.s^?1 .(g. dry wt.)^?1. From the rate of ATP synthesis and the separately measured oxygen consumption we calculated an apparent mitochondrial ADP:O ratio of 3.5 ± 0.8 in the intact tissue.     

Modeling the energy transfer pathways. creatine kinase activities and heterogeneous distribution of ADP in the perfused heart

The exchange scheme of high energy phosphate transport in a whole heart relies on a system of CK functioning in different ways. This suggests that the CKs are able to act both like a shuttle and like a buffer for the energy transfer. The challenge is to understand how these two functions are balanced in the CK system. One key of this balance is the knowledge of the local concentrations of the ADP nucleotide. These concentrations cannot be directly measured, but they may be derived by computation. In the present report we introduce the known properties of the enzymes catalyzing the exchange of high energy phosphate into the model of flux pathways derived from NMR experiments to compute both the maximum activity of each enzyme and the local concentrations of all the substrates. We show that the ADP distribution must be heterogeneous for the system to work. Its concentration is 50% higher in the vicinity of ATPase sites and 50% lower in the intermembrane space of the mitochondria than in the cytosol. Another result of this analysis is that the apparent large unbalance of the CKmito pathway is imposed by the adenosine nucleotide transferase fluxes. This analysis proves that it is possible to deduce the local concentrations of a substrate by combining data originating from NMR, biochemistry and enzymology into a common model.     

Adrenergic stimulation of the heart during inhibition of phosphocreatine or adenylate pathways of energy transfer in cardiomyocytes]

Functional and metabolic response of an isovolumically perfused heart of a rat to isoproterenol (0.1 microM) has been studied. A heart with the normal content of adenine nucleotides (AN) and phosphocreatine (PCr) as well as that with the 5-fold reduced AN content (with 2-deoxyglucose treatment) significantly increased cardiac work index (PRP), maximal contraction rate (MCR) and maximal relaxation rate (MRR) (by 50, 30-40 and 100-150%, respectively). The effect was preserved for all the period of the hormone action (30 min) and was followed by a temporary decrease in the PCr content. The heart with an inhibited unidirectional flux of metabolites through creative kinase (CK) and normal level of AN responded to the hormone by the slower and decelerated growth of the function and in the heart with almost completely iodoacetamide (IAAm)-blocked CK the functional response was minimal and transient. In the latter a significant and irreversible decline in PCr and ATP content and a concomitant rise of inorganic phosphate took place. Both basal and isoproterenol-stimulated adenylate cyclase activity remained unchanged after IAAm treatment. An increase in PRP correlated with the elevation of the cytosolic ADP concentration, however, correlation was not uniform for different experimental groups. These data show significance of the creatine kinase system not only for maintenance of maximal work but also for a rapid functional response to the catecholamine stimulation.     

NMR indirect detection of glutamate to measure citric acid cycle flux in the isolated perfused mouse heart

(13)C-edited proton nuclear magnetic resonance (NMR) spectroscopy was used to follow enrichment of glutamate C3 and C4 with a temporal resolution of approximately 20 s in mouse hearts perfused with (13)C-enriched substrates. A fit of the NMR data to a kinetic model of the tricarboxylic acid (TCA) cycle and related exchange reactions yielded TCA cycle (V(tca)) and exchange (V(x)) fluxes between alpha-ketoglutarate and glutamate. These fluxes were substrate-dependent and decreased in the order acetate (V(tca)=14.1 micromol g(-1) min(-1); V(x)=26.5 micromol g(-1) min(-1))>octanoate (V(tca)=6.0 micromol g(-1) min(-1); V(x)=16.1 micromol g(-1) min(-1))>lactate (V(tca)=4.2 micromol g(-1) min(-1); V(x)=6.3 micromol g(-1) min(-1)).     

NMR observability of ATP: preferential depletion of cytosolic ATP during ischemia in perfused rat liver

The extent to which cellular metabolites are NMR observable is of fundamental importance in the interpretation of in vivo NMR studies. Analysis of ischemic rat liver shows that ATP resonances measured by 31P NMR decrease considerably faster than total tissue ATP measured in extracts. This discrepancy demonstrates that, in liver, ATP is not 100% observable. Furthermore, the data are consistent with the supposition that in situ mitochondrial ATP resonances are not normally observable by in vivo NMR techniques. The specificity of the NMR measurement for cytosolic ATP indicates that 31P NMR can be a valuable tool for the specific measurement of ATP in this compartment.     

Regulation of energy flux through the creatine kinase reaction in vitro and in perfused rat heart: P-NMR studies

Fluxes catalyzed by soluble creatine kinase (MM) in equilibrium in vitro and by the creatine kinase system in perfused rat hearts were studied by ³¹P-NMR saturation transfer method. It was found that in vitro both forward and reverse fluxes through creatine kinase at equilibrium were almost equal and very stable to changes in ratio (from 0.2 to 3.0) as well as to changes in pH (from 7.4 to 6.5 or 8.1), free Mg²⁺ concentration and 2-fold decrease of total adenine nucleotides and creatine pools (from 8.0 to 4.0 mM and from 30 to 14 mM, respectively). In the rat hearts perfused by the Langendorff method the creatine kinase-catalyzed flux from phosphocreatine to ATP was increased by 50% when oxygen consumption grew from 8 to 55 μmol/min per g of dry wt. due to transition from rest to high workload. These changes could not be exclusively explained on the basis of the equilibrium model by activation of heart creatine kinase due to some decrease in ratio (from 1.8 to 0.8) observed during transition from rest to high workload. Analysis of our data showed that an increase in the flux via creatine kinase is correlated with an increase in the rate of ATP synthesis with a linearity coefficient higher than 1.0. These data are more consistent with the concept of energy channeling by phosphocreatine shuttle than with that of the creatine kinase equilibrium in the heart.     

Regulation of energy flux through the creatine kinase reaction in vitro and in perfused rat heart: 31P-NMR studies

Fluxes catalyzed by soluble creatine kinase (MM) in equilibrium in vitro and by the creatine kinase system in perfused rat hearts were studied by ³¹P-NMR saturation transfer method. It was found that in vitro both forward and reverse fluxes through creatine kinase at equilibrium were almost equal and very stable to changes in $\text{phosphocreatine}\text{creatine}$ ratio (from 0.2 to 3.0) as well as to changes in pH (from 7.4 to 6.5 or 8.1), free Mg²⁺ concentration and 2-fold decrease of total adenine nucleotides and creatine pools (from 8.0 to 4.0 mM and from 30 to 14 mM, respectively). In the rat hearts perfused by the Langendorff method the creatine kinase-catalyzed flux from phosphocreatine to ATP was increased by 50% when oxygen consumption grew from 8 to 55 μmol/min per g of dry wt. due to transition from rest to high workload. These changes could not be exclusively explained on the basis of the equilibrium model by activation of heart creatine kinase due to some decrease in $\text{[}\text{phosphocreatine}\text{]}\text{[}\text{creatine}\text{]}$ ratio (from 1.8 to 0.8) observed during transition from rest to high workload. Analysis of our data showed that an increase in the flux via creatine kinase is correlated with an increase in the rate of ATP synthesis with a linearity coefficient higher than 1.0. These data are more consistent with the concept of energy channeling by phosphocreatine shuttle than with that of the creatine kinase equilibrium in the heart.     

Multinuclear NMR studies of the Langendorff perfused rat heart

The quantitation of intracellular sodium ion concentration [Na+]in perfused organs using NMR spectroscopy requires a knowledge of the extent of visibility of the 23Na resonance and of the intracellular volume of the organ. We have used a multinuclear NMR approach, in combination with the extracellular shift reagent dysprosium (III) tripolyphosphate, to determine the NMR visibility of intra- and extracellular 23Na and 35Cl ions, intracellular volume, and [Na+]in in the isolated Langendorff perfused rat heart. Based on a comparison of the extracellular volumes calculated using 2H and 23Na, 35Cl, or 59Co NMR of the perfused heart we conclude that resonances of extracellular sodium and chloride ions (including ions in interstitial spaces) are fully visible, contrary to assumptions in the literature. Furthermore, prolonged hypoxia or ischemia caused a dramatic increase in intracellular Na+ and [Na+] in rose to approach that in the external medium indicating full visibility of the intracellular 23Na resonance. Resonance intensities of intra- and extracellular 23Na ions, along with a knowledge of the extracellular space as a fraction of the total organ water space, yielded an average [Na+] in of about 10 mM (10 +/- 1.5 mM) for the rat heart at 37 degrees C. Double-quantum filtered 23Na NMR of the perfused rat heart in the absence and presence of paramagnetic reagents revealed, contrary to assumptions in the literature, that both intra- and extracellular sodium ions contribute to the detected signal.     

Lipid-mediated impairment of normal energy metabolism in the isolated perfused diabetic rat heart studied by phosphorus-31 NMR and chemical extraction

The relationship between extracellular palmitate and the accumulation of long-chain fatty-acyl coenzyme A with that of high-energy phosphate metabolism was investigated in the isolated perfused diabetic rat heart. Hearts were perfused with a glucose/albumin buffer supplemented with 0, 0.5, 1.2 or 2.0 mM palmitate. ³¹P-NMR was used to analyze phosphocreatine and ATP metabolism during 1 h of constant-flow recirculation perfusion. At the end of perfusion, frozen samples were taken for chemical analysis of high-energy phosphates and the free and acylated fractions of coenzyme A and carnitine. Perfusion of diabetic hearts with palmitate, unlike control hearts, caused a time-dependent and concentration-dependent reduction in ATP, despite normal and constant phosphocreatine. Concentrations of acid-soluble coenzyme A, long-chain-acyl coenzyme A and total tissue coenzyme A were elevated in palmitate-perfused diabetic hearts, while the total tissue carnitine pool was decreased. Increases in long-chain-acyl coenzyme A correlated with the reduction in myocardial ATP. This reduction in ATP could not be adequately explained by alterations in heart rate, perfusion pressure or vascular resistance.     

Regulation of energy flux through the creatine kinase reaction in vitro and in perfused rat heart. 31P-NMR studies

Fluxes catalyzed by soluble creatine kinase (MM) in equilibrium in vitro and by the creatine kinase system in perfused rat hearts were studied by 31P-NMR saturation transfer method. It was found that in vitro both forward and reverse fluxes through creatine kinase at equilibrium were almost equal and very stable to changes in phosphocreatine/creatine ratio (from 0.2 to 3.0) as well as to changes in pH (from 7.4 to 6.5 or 8.1), free Mg2+ concentration and 2-fold decrease of total adenine nucleotides and creatine pools (from 8.0 to 4.0 mM and from 30 to 14 mM, respectively). In the rat hearts perfused by the Langendorff method the creatine kinase-catalyzed flux from phosphocreatine to ATP was increased by 50% when oxygen consumption grew from 8 to 55 mumol/min per g of dry wt. due to transition from rest to high workload. These changes could not be exclusively explained on the basis of the equilibrium model by activation of heart creatine kinase due to some decrease in [phosphocreatine]/[creatine] ratio (from 1.8 to 0.8) observed during transition from rest to high workload. Analysis of our data showed that an increase in the flux via creatine kinase is correlated with an increase in the rate of ATP synthesis with a linearity coefficient higher than 1.0. These data are more consistent with the concept of energy channeling by phosphocreatine shuttle than with that of the creatine kinase equilibrium in the heart.     

Evidence of separate pathways for lactate uptake and release by the perfused rat heart

The simultaneous release and uptake of lactate by the heart has been observed both in vivo and ex vivo; however, the pathways underlying these observations have not been satisfactorily explained. Consequently, the purpose of this study was to test the hypothesis that hearts release lactate from glycolysis while simultaneously taking up exogenous lactate. Therefore, we determined the effects of fatty acids and diabetes on the regulation of lactate uptake and release. Hearts from control and 1-wk diabetic animals were perfused with 5 mM glucose, 0.5 mM [3-(13)C]lactate, and 0, 0.1, 0.32, or 1.0 mM palmitate. Parameters measured include perfusate lactate concentrations, fractional enrichment, and coronary flow rates, which enabled the simultaneous, but independent, measurements of the rates of 1) uptake of exogenous [(13)C]lactate and 2) efflux of unlabeled lactate from metabolism of glucose. Although the rates of lactate uptake and efflux were both similarly inhibited by the addition of palmitate, (i.e., the ratio of lactate uptake to efflux remained constant), the ratio of lactate uptake to efflux was significantly higher in the controls compared with the diabetic group (1.00 +/- 0.14 vs. 0.50 +/- 0.07, P < 0.002). These data, combined with heterogeneous (13)C enrichment of tissue lactate, pyruvate, and alanine, suggest that glycolytically derived lactate production and oxidation of exogenous lactate operate as functionally separate metabolic pathways. These results are consistent with the concept of an intracellular lactate shuttle.     

Evaluation of carbon flux and substrate selection through alternate pathways involving the citric acid cycle of the heart by 13C NMR spectroscopy

A previous 13C NMR technique (Malloy, C. R., Sherry, A.D., and Jeffrey, F.M.H. (1987) FEBS Lett. 212, 58-62) for measuring the relative flux of molecules through the oxidative versus anaplerotic pathways involving the citric acid cycle of the rat heart has been extended to include a complete analysis of the entire glutamate 13C spectrum. Although still simple in practice, this more sophisticated model allows an evaluation of 13C fractional enrichment of molecules entering both the oxidative and anaplerotic pathways under steady-state conditions. The method was used to analyze 13C NMR spectra of intact hearts or their acid extracts during utilization of 13C-enriched pyruvate, propionate, acetate, or various combinations thereof. [2-13C]Pyruvate was used to prove that steady-state flux of pyruvate through pyruvate carboxylase is significant during co-perfusion of pyruvate and acetate, and we demonstrate for the first time that a nine-line 13C multiplet may be detected in an intact, beating heart. Acetate or pyruvate alone provided about 86% of the acetyl-CoA; in combination, about 65% of the acetyl-CoA was derived from acetate, about 30% was derived from pyruvate, and the remainder from endogenous sources. Propionate reduced the contribution of exogenous acetate to acetyl-CoA to 77% and also reduced the oxidation of endogenous substrates. Equations are presented which allow this same analysis on multiply labeled substrates, making this technique extremely powerful for the evaluation of substrate selection and relative metabolic flux through anaplerotic and oxidative pathways in the intact heart.     

ATP synthesis and degradation rates in the perfused rat heart. 31P-nuclear magnetic resonance double saturation transfer measurements.

A limitation of magnetization transfer techniques for studying enzyme kinetics in vivo has been the difficulty of treating systems with more than two exchanging species. This problem was addressed in the original papers describing saturation transfer. Since then, a number of approaches have been devised to study these complex situations. Here, we present a method based on the transient saturation transfer experiment in which spin-lattice relaxation time constants and reaction rates are obtained from the same magnetization transfer data. This technique is particularly suitable for biological samples. We apply the method to evaluate flux balance in the three-site linear exchange network composed of ATP, creatine phosphate, and inorganic phosphate in the isolated, perfused rat heart and show that the method yields reasonable values for the reaction velocities of ATP synthesis and degradation.     

31P NMR measurement of ATP synthesis rate in perfused intact rat hearts

Using 31P NMR and the saturation-transfer method, the unidirectional rate of ATP synthesis was measured in isolated, Langendorff-perfused, isovolumic rat hearts operating at a rate pressure product of 25.6 +/- 2.5 (SE) X 10(3) mmHg X min-1 and consuming O2 at a rate of 35 +/- 2 mumol O2 X min-1 X (g dry wt)-1, at 37 degrees C. This rate was 7.2 +/- 0.9 mumol X s-1 X (g dry wt)-1 and was related to the rate of oxygen atom consumption by a ratio of 6.3 +/- 0.9. These data show that in the intact heart the unidirectional rate of ATP synthesis exceeds the net rate of ATP synthesis and consumption by approximately a factor of 2.