Modeling non‐linear kinetics of hyperpolarized [1‐13C] pyruvate in the crystalloid‐perfused rat heart

Hyperpolarized ¹³C MR measurements have the potential to display non‐linear kinetics. We have developed an approach to describe possible non‐first‐order kinetics of hyperpolarized [1‐¹³C] pyruvate employing a system of differential equations that agrees with the principle of conservation of mass of the hyperpolarized signal. Simultaneous fitting to a second‐order model for conversion of [1‐¹³C] pyruvate to bicarbonate, lactate and alanine was well described in the isolated rat heart perfused with Krebs buffer containing glucose as sole energy substrate, or glucose supplemented with pyruvate. Second‐order modeling yielded significantly improved fits of pyruvate–bicarbonate kinetics compared with the more traditionally used first‐order model and suggested time‐dependent decreases in pyruvate–bicarbonate flux. Second‐order modeling gave time‐dependent changes in forward and reverse reaction kinetics of pyruvate–lactate exchange and pyruvate–alanine exchange in both groups of hearts during the infusion of pyruvate; however, the fits were not significantly improved with respect to a traditional first‐order model. The mechanism giving rise to second‐order pyruvate dehydrogenase (PDH) kinetics was explored experimentally using surface fluorescence measurements of nicotinamide adenine dinucleotide reduced form (NADH) performed under the same conditions, demonstrating a significant increase of NADH during pyruvate infusion. This suggests a simultaneous depletion of available mitochondrial NAD⁺ (the cofactor for PDH), consistent with the non‐linear nature of the kinetics. NADH levels returned to baseline following cessation of the pyruvate infusion, suggesting this to be a transient effect. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.     

Application of hyperpolarized [1-(13) C]lactate for the in vivo investigation of cardiac metabolism

In addition to cancer imaging, ¹³C‐MRS of hyperpolarized pyruvate has also demonstrated utility for the investigation of cardiac metabolism and ischemic heart disease. Although no adverse effects have yet been reported for doses commonly used in vivo, high substrate concentrations have lead to supraphysiological pyruvate levels that can affect the underlying metabolism and should be considered when interpreting results. With lactate serving as an important energy source for the heart and physiological lactate levels one to two orders of magnitude higher than for pyruvate, hyperpolarized lactate could potentially be used as an alternative to pyruvate for probing cardiac metabolism. In this study, hyperpolarized [1‐¹³C]lactate was used to acquire time‐resolved spectra from the healthy rat heart in vivo and to measure dichloroacetate (DCA)‐modulated changes in flux through pyruvate dehydrogenase (PDH). Both primary oxidation of lactate to pyruvate and subsequent conversion of pyruvate to alanine and bicarbonate could reliably be detected. Since DCA stimulates the activity of PDH through inhibition of PDH kinase, a more than 2.5‐fold increase in bicarbonate‐to‐substrate ratio was found after administration of DCA, similar to the effect when using [1‐¹³C]pyruvate as the substrate. Copyright © 2012 John Wiley & Sons, Ltd.     

Functional and biochemical effects of a K+-ionophore on the isolated perfused rat heart

Valinomycin, a K⁺-specific ionophore, influenced function and metabolism of isolated, perfused rat hearts in a dose-dependent fashion. At a concentration of 0.05 μg ml^-1 in perfusion fluid a 50% reduction of heart rate (HR) and a 90% reduction in max dP/dt were observed. These effects were paralleled by a substantial decrease of myocardial energy charge from about 0.80 to 0.20. A 2.5 fold increase in tissue lactate concentration indicated an increased rate of glycolytic activity. Low ATP combined with high ADP and AMP levels as found in these valinomycin-treated hearts is known to promote phosphofructokinase activity and may explain the elevated lactate levels. A significant increase in the concentrations of adenosine, IMP and inosine was observed as well.     

In vivo detection of d‐amino acid oxidase with hyperpolarized d‐[1‐13C]alanine

d ‐amino acid oxidase (DAO) is a peroxisomal enzyme that catalyzes the oxidative deamination of several neutral and basic d ‐amino acids to their corresponding α‐keto acids. In most mammalian species studied, high DAO activity is found in the kidney, liver, brain and polymorphonuclear leukocytes, and its main function is to maintain low circulating d ‐amino acid levels. DAO expression and activity have been associated with acute and chronic kidney diseases and with several pathologies related to N‐methyl‐d ‐aspartate (NMDA) receptor hypo/hyper‐function; however, its precise role is not completely understood. In the present study we show that DAO activity can be detected in vivo in the rat kidney using hyperpolarized d ‐[1‐¹³C]alanine. Following a bolus of hyperpolarized d ‐alanine, accumulation of pyruvate, lactate and bicarbonate was observed only when DAO activity was not inhibited. The measured lactate‐to‐d ‐alanine ratio was comparable to the values measured when the l ‐enantiomer was injected. Metabolites downstream of DAO were not observed when scanning the liver and brain. The conversion of hyperpolarized d ‐[1‐¹³C]alanine to lactate and pyruvate was detected in blood ex vivo, and lactate and bicarbonate were detected on scanning the blood pool in the heart in vivo; however, the bicarbonate‐to‐d ‐alanine ratio was significantly lower compared with the kidney. These results demonstrate that the specific metabolism of the two enantiomers of hyperpolarized [1‐¹³C]alanine in the kidney and in the blood can be distinguished, underscoring the potential of d ‐[1‐¹³C]alanine as a probe of d ‐amino acid metabolism.     

Validation of the in vivo assessment of pyruvate dehydrogenase activity using hyperpolarised 13C MRS

Many diseases of the heart are characterised by changes in substrate utilisation, which is regulated in part by the activity of the enzyme pyruvate dehydrogenase (PDH). Consequently, there is much interest in the in vivo evaluation of PDH activity in a range of physiological and pathological states to obtain information on the metabolic mechanisms of cardiac diseases. Hyperpolarised [1‐¹³C]pyruvate, detected using MRS, is a novel technique for the noninvasive evaluation of PDH flux. PDH flux has been assumed to directly reflect in vivo PDH activity, although to date this assumption remains unproven. Control animals and animals undergoing interventions known to modulate PDH activity, namely high fat feeding and dichloroacetate infusion, were used to investigate the relationship between in vivo hyperpolarised MRS measurements of PDH flux and ex vivo measurements of PDH enzyme activity (PDH_a). Further, the plasma concentrations of pyruvate and other important metabolites were evaluated following pyruvate infusion to assess the metabolic consequences of pyruvate infusion during hyperpolarised MRS experiments. Hyperpolarised MRS measurements of PDH flux correlated significantly with ex vivo measurements of PDH_a, confirming that PDH activity influences directly the in vivo flux of hyperpolarised pyruvate through cardiac PDH. The maximum plasma concentration of pyruvate reached during hyperpolarised MRS experiments was approximately 250 µM , equivalent to physiological pyruvate concentrations reached during exercise or with dietary interventions. The concentrations of other metabolites, including lactate, glucose and β‐hydroxybutyrate, did not vary during the 60 s following pyruvate infusion. Hence, during the 60‐s data acquisition period, metabolism was minimally affected by pyruvate infusion. Copyright © 2010 John Wiley & Sons, Ltd.     

Accelerating hyperpolarized metabolic imaging of the heart by exploiting spatiotemporal correlations

Hyperpolarized ¹³C‐labeled pyruvate is a promising tool to investigate cardiac metabolism. It has been shown that changes in substrate metabolism occur following the induction of ischemia. To investigate the metabolic changes that are confined to spatial regions, high spatiotemporal resolution is required. The present work exploits both spatial and temporal correlations using k–t principal component analysis (PCA) to undersample the spatiotemporal domain, thereby speeding up data acquisition. A numerical model was implemented to investigate optimal acquisition and reconstruction parameters for pyruvate, lactate and bicarbonate maps of the heart. Subsequently, prospectively undersampled in vivo data on rat hearts were acquired using a combination of spectral–spatial signal excitation and a variable‐density single‐shot echo planar readout. Using five‐fold k–t PCA, a spatial resolution of 1 × 1 mm² at a temporal resolution of 3 s was achieved. Copyright © 2013 John Wiley & Sons, Ltd.     

Regulation der Pyruvatdehydrogenaseaktivität und Dynamik des isoliert perfundierten Meerschweinchenherzens im Thiaminmangel

Summary Isolated hearts of guinea pigs were perfused according to Langendorff's method with pyruvate-1-¹⁴C as substrate. The following criteria of the heart function were measured: frequence, left ventricular pressure, the rate of change of left intraventricular pressure (dp/dt), and isovolumetric work and as parameters of the metabolism the uptake of pyruvate and oxygen and the output of lactate. The effect of epinephrine on the above mentioned parameters was measured in the hearts of normal and thiamine deficient animals. Oxythiamine diminished significantly all functional and metabolic parameters in normal hearts. The rise of the functional and metabolic parameters by epinephrine was significantly diminished in the normal hearts when pretreated with oxythiamine. The heart rate was hardly enhanced by epinephrine in the hearts of thiamine deficient animals. The left ventricular pressure,dp/dt, isovolumetric work and oxygen consumption were only slightly raised. While the metabolism of pyruvate was elevated in normal guinea pig hearts by epinephrine up to 72%, there was only an enhancement of about 28% in dietary thiamine deficiency. Oxythiamine diminished the pyruvate metabolism in oxythiamine treated hearts by –36%. These findings suggest that the regulation of the activity of the pyruvate dehydrogenase is already limited in the early stages of thiamine deficiency. A dissociation between the uptake of substrate and the mechanical parameters of the heart could be demonstrated in the hearts of thiamine deficient animals during the application of epinephrine.

Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Substrate selection in hearts subjected to ischemia/reperfusion: role of cardioplegic solutions and gender

In conditions of ischemia/reperfusion (I/R), the relative use of all available substrates by the heart has a significant effect on the recovery of the organ. This substrate preference in perfused hearts is influenced by ischemia. We followed the metabolic fate of [U‐¹³C]glucose and [3‐¹³C]lactate in hearts preserved in Celsior (Cs) and histidine buffer solution (HBS) for 4 or 6 h and subsequently perfused with a Krebs–Henseleit solution (KH) containing [U‐¹³C]glucose and [3‐¹³C]lactate. We also assessed gender‐specific metabolic modulation in our I/R experimental conditions. Hearts from male and female Wistar rats (6–8 weeks) were subjected to moderate (0–240 min) or prolonged (240–360 min) cold ischemia whilst immersed in Cs and HBS, and perfused for 30 min with KH containing [U‐¹³C]glucose and [3‐¹³C]lactate. After perfusion, hearts were freeze‐clamped and metabolites were extracted for ¹³C NMR isotopomer analysis. In control conditions, there were no differences with regard to lactate origin in hearts from males and females. After 6 h of preservation in Cs, lactate origin was mostly from [U‐¹³C]glucose in hearts from males and from [3‐¹³C]lactate in hearts from females. During the 6 h of organ preservation in HBS, the lactate pool showed a strong contribution from unenriched sources in male hearts and from [U‐¹³C]glucose in female hearts. The glutamate C2/C4 ratio was stable or increased in hearts from females after I/R, and the alanine index increased in hearts from both males and females. Octanoate was, as predicted, the preferential substrate during perfusion. Glucose and lactate suffer a distinct metabolic fate in our I/R conditions, which is related to the cardioplegic solution used during organ storage, and the gender. Hearts from females appear to be less sensitive to I/R injury, and heart preservation in HBS proved to be effective in enhancing anaplerosis during perfusion, especially in hearts from females. Copyright © 2011 John Wiley & Sons, Ltd.     

Increase of fibronectin and osteopontin in porcine hearts following ischemia and reperfusion

Following a severe ischemic injury or myocardial infarction, the extracellular matrix (ECM) of the heart is involved in pathophysiological conditions such as dilatation and cardiac dysfunction. Osteopontin (OPN) has been shown to interact with fibronectin suggesting its possible role in matrix organization, stability and wound healing. There is increased expression of OPN in several tissues in response to injury. Therefore, we tested the hypothesis that acute ischemia (2 h), followed by reperfusion (4 h) may induce early OPN and fibronectin in an isolated hemoperfused working porcine heart model. Twenty hearts were prepared and connected to a perfusion system. After 1 h of perfusion, these hearts were randomized to two groups: ten infarcted (MI, ramus circumflexus) and ten non-infarcted hearts (C). In addition, cardiac fibroblasts derived from infarcted, remote and control myocardium were investigated. In both groups, the heart rate, electrolytes, pH, blood gases, and lactate remained similar. The LVEDP and perfusion pressure of MI hearts increased significantly (P<0.05). The total fibronectin and OPN volume contents were clearly elevated in the infarct area. The matrix metalloproteinases (MMP-1 and MMP-8), fibronectin, OPN, TGF-₁ proteins and the mRNAs for fibronectin, TGF-₁, and OPN were significantly elevated in the infarct area as compared to the remote area and the non-infarcted hearts. Simultaneously, circulating carboxyterminal propeptide of type I procollagen (PICP) was released in the perfusion medium (threefold versus C). Fibroblast-like cells originating from the infarct area exhibited an enhanced OPN and fibronectin gene and protein expression compared to fibroblasts derived from control myocardium. Our data demonstrate the early appearance of the MMPs (increased collagen degrading enzymes) and PICP (a collagen synthesis marker) following ischemia and reperfusion. Moreover, OPN, fibronectin and TGF-₁ protein and gene expression are elevated after ischemia and reperfusion in the ex vivo working hemoperfused porcine heart model.This research was supported by the Deutsche Forschungsgemeinschaft (DFG) grants GR 1039/7-1 and GR 1039/7-2 to D.G.     

Metabolism of hyperpolarized [1-(13) C]pyruvate in the isolated perfused rat lung - an ischemia study

We report studies of the effect of ischemia on the metabolic activity of the intact perfused lung and its restoration after a period of reperfusion. Two groups of rat lungs were studied using hyperpolarized 1‐¹³C pyruvate to compare the rate of lactate labeling differing only in the temporal ordering of ischemic and normoxic acquisitions. In both cases, a several‐fold increase in lactate labeling was observed immediately after a 25‐min ischemia event as was its reversal back to the baseline after 30–40 min of resumed perfusion (n = 5, p < 0.025 for both comparisons). These results were corroborated by ³¹P spectroscopy and correspond well to measured changes in lactate pool size determined by ¹H spectroscopy of freeze‐clamped specimens. Copyright © 2012 John Wiley & Sons, Ltd.     

Carbachol-induced increase in inositol trisphosphate (IP3) content is attenuated by adrenergic stimulation in the isolated working rat heart

The interrelated responses of concomitant adrenergic and muscarinic receptor stimulation on second messengers and mechanical activity in the isolated perfused working rat heart were studied. The hearts were perfused with Krebs-Henseleit buffer in a modified Langendorff apparatus. The hearts were perfused with noradrenaline (10^-6 mol L^-1, n= 20), with carbachol (3 times 10^-7 mol L^-1n= 11) or with noradrenaline plus carbachol (n= 20) in the above-mentioned concentrations. The hearts were frozen at 20 s, 30 s and 40 min after addition of noradrenaline and noradrenaline plus carbachol and at 20 s and 40 min after addition of carbachol. Five hearts were freeze-clamped directly after preperfusion and another five hearts after 40 min of perfusion and used as controls. Myocardial cAMP increased at 20 s and 40 min after noradrenaline perfusion. In contrast to this cAMP was unchanged at 20 s and decreased at 40 min after perfusion with noradrenaline plus carbachol. IP₃ content increased after 20 s of carbachol- and after 40 min of noradrenaline perfusion (P < 0.05). However, noradrenaline plus carbachol did not induced any significant increase in IP₃ content after 20 s and 30 s, but after 40 min a decrease below basal level was found (P < 0.05). Noradrenaline stimulation attenuated muscarinic agonist induced IP₃ formation. A reciprocity existed in that noradrenaline induced IP₃ formation was attenuated by carbachol. No direct relationship was observed between the IP₃ response and contractility, also valid for cAMP. Hence, a significant increase in contractility was found in spite of a blunted cAMP response probably not accounted for by an an α₁-adrenergic effect alone.     

Probing lactate secretion in tumours with hyperpolarised NMR

Most tumours exhibit a high rate of glycolysis and predominantly produce energy by lactic acid fermentation. To maintain energy production and prevent toxicity, the lactate generated needs to be rapidly transported out of the cell. This is achieved by monocarboxylate transporters (MCTs), which therefore play an essential role in cancer metabolism and development. In vivo experiments were performed on eight male Fisher F344 rats bearing a subcutaneous mammary carcinoma after injection of hyperpolarised [1‐¹³C]pyruvate. A Gd(III)DO3A complex that binds to pyruvate and its metabolites was used to efficiently destroy the extracellular magnetisation after hyperpolarised lactate had been formed. Moreover, a pulse sequence including a frequency‐selective saturation pulse was designed so that the pyruvate magnetisation could be destroyed to exclude effects arising from further conversion. Given this preparation, metabolite transport out of the cell manifested as additional decay and apparent cell membrane transporter rates could thus be obtained using a reference measurement without a relaxation agent. In addition to slice‐selective spectra, spatially resolved maps of apparent membrane transporter activity were acquired using a single‐shot spiral gradient readout. A considerable increase in decay rate was detected for lactate, indicating rapid transport out of the cell. The alanine signal was unaltered, which corresponds to a slower efflux rate. This technique could allow for better understanding of tumour metabolism and progression, and enable treatment response measurements for MCT‐targeted cancer therapies. Moreover, it provides vital insights into the signal kinetics of hyperpolarised [1‐¹³C]pyruvate examinations. Copyright © 2016 John Wiley & Sons, Ltd.     

Kinetic modelling of dissolution dynamic nuclear polarisation 13C magnetic resonance spectroscopy data for analysis of pyruvate delivery and fate in tumours

Dissolution dynamic nuclear polarisation (dDNP) of ¹³C-labelled pyruvate in magnetic resonance spectroscopy/imaging (MRS/MRSI) has the potential for monitoring tumour progression and treatment response. Pyruvate delivery, its metabolism to lactate and efflux were investigated in rat P22 sarcomas following simultaneous intravenous administration of hyperpolarised ¹³C-labelled pyruvate (¹³C₁-pyruvate) and urea (¹³C-urea), a nonmetabolised marker. A general mathematical model of pyruvate-lactate exchange, incorporating an arterial input function (AIF), enabled the losses of pyruvate and lactate from tumour to be estimated, in addition to the clearance rate of pyruvate signal from blood into tumour, K_ip, and the forward and reverse fractional rate constants for pyruvate-lactate signal exchange, k_pl and k_lp. An analogous model was developed for urea, enabling estimation of urea tumour losses and the blood clearance parameter, K_iu. A spectral fitting procedure to blood time-course data proved superior to assuming a gamma-variate form for the AIFs. Mean arterial blood pressure marginally correlated with clearance rates. K_iu equalled K_ip, indicating equivalent permeability of the tumour vasculature to urea and pyruvate. Fractional loss rate constants due to effluxes of pyruvate, lactate and urea from tumour tissue into blood (k_po, k_lo and k_uo, respectively) indicated that T₁s and the average flip angle, θ, obtained from arterial blood were poor surrogates for these parameters in tumour tissue. A precursor-product model, using the tumour pyruvate signal time-course as the input for the corresponding lactate signal time-course, was modified to account for the observed delay between them. The corresponding fractional rate constant, k_avail, most likely reflected heterogeneous tumour microcirculation. Loss parameters, estimated from this model with different TRs, provided a lower limit on the estimates of tumour T₁ for lactate and urea. The results do not support use of hyperpolarised urea for providing information on the tumour microcirculation over and above what can be obtained from pyruvate alone. The results also highlight the need for rigorous processes controlling signal quantitation, if absolute estimations of biological parameters are required.     

Dynamic changes of myocardial inositoltrisphosphate and cyclic nucleotides: relationship to contractile response in the perfused working rat heart after adrenergic and muscarinic agonist stimulation

Initial and late effects by adrenergic and muscarinic agonists on inositol (1,4,5) trisphosphate (IP₃) and cyclic nucleotide levels were determined and correlated to mechanical response in perfused rat hearts. Forty-three rat hearts were perfused with Krebs-Henseleit buffer in a modified Langendorff apparatus as a working preparation. The hearts were perfused as controls (n= 11), or with noradrenaline (10^-6 mol 1^-1) (n= 21), or with carbachol (3 × 10^-7 mol 1^-1) (n= 11) added to the perfusion buffer. The hearts were frozen at 20 s, 30 s and 40 min after addition of noradrenaline and at 20 s and 40 min after addition of carbachol, and after 5 and 45 min of control perfusion. cAMP and cGMP were determined by radioligand methods and IP₃ by a combined fast performance liquid chromatography (FPLC) - isotachophoretic method. cAMP increased by 36% within 20 s followed by a decrease (22%) during the 10 s following noradrenaline addition. After 40 min cAMP regained its value near that of 20 s. Noradrenaline perfusion did not influence IP₃ levels during the first 30 s although the value at 40 min was significantly higher (59%). IP₃ increased (42%) after 20 s of carbachol perfusion followed by a 25 % decrease at 40 min. Sustained stimulation of β-receptors (after 40 min in our model) resulted in a repeated increase in cAMP only, without an increase in contractility. Muscarinic receptor stimulation resulted in an early increase in IP₃ content without a change in mechanical function whereas a-adrenergic stimulation resulted in only a late increase in IP₃ content of isolated whole hearts still without any alteration in contractility.     

Assessing the effect of hypoxia on cardiac metabolism using hyperpolarized 13C magnetic resonance spectroscopy

Hypoxia plays a role in many diseases and can have a wide range of effects on cardiac metabolism depending on the extent of the hypoxic insult. Noninvasive imaging methods could shed valuable light on the metabolic effects of hypoxia on the heart in vivo. Hyperpolarized carbon‐13 magnetic resonance spectroscopy (HP ¹³C MRS) in particular is an exciting technique for imaging metabolism that could provide such information. The aim of our work was, therefore, to establish whether hyperpolarized ¹³C MRS can be used to assess the in vivo heart's metabolism of pyruvate in response to systemic acute and chronic hypoxic exposure. Groups of healthy male Wistar rats were exposed to either acute (30 minutes), 1 week or 3 weeks of hypoxia. In vivo MRS of hyperpolarized [1‐¹³C] pyruvate was carried out along with assessments of physiological parameters and ejection fraction. Hematocrit was elevated after 1 week and 3 weeks of hypoxia. 30 minutes of hypoxia resulted in a significant reduction in pyruvate dehydrogenase (PDH) flux, whereas 1 or 3 weeks of hypoxia resulted in a PDH flux that was not different to normoxic animals. Conversion of hyperpolarized [1‐¹³C] pyruvate into [1‐¹³C] lactate was elevated following acute hypoxia, suggestive of enhanced anaerobic glycolysis. Elevated HP pyruvate to lactate conversion was also seen at the one week timepoint, in concert with an increase in lactate dehydrogenase (LDH) expression. Following three weeks of hypoxic exposure, cardiac metabolism of pyruvate was comparable with that observed in normoxia. We have successfully visualized the effects of systemic hypoxia on cardiac metabolism of pyruvate using hyperpolarized ¹³C MRS, with differences observed following 30 minutes and 1 week of hypoxia. This demonstrates the potential of in vivo hyperpolarized ¹³C MRS data for assessing the cardiometabolic effects of hypoxia in disease.     

Effects of noradrenaline and flow on lactate uptake in the perfused rat hindlimb

Skeletal muscle can release or take up lactate depending on the lactate concentration gradient across the cell membrane. In the perfused rat hindlimb without arterial lactate, both noradrenaline (NA) infusion and increased flow promote lactate release and oxygen consumption (VO ₂). However, it is unclear whether NA or increased flow rate have similar effects on lactate uptake. The present study compares these effects in the rat hindlimb perfused at a basal flow rate of 0.33 mL min^?1 g^?1 and 25 °C in the presence of added arterial lactate. When 10 mmol L^?1 L-(+)-lactate was added to the arterial perfusate, lactate was taken up (16 ± 1.0 μmol g^?1 h^?1, n = 13) by the hindlimb with a 35% higher VO ₂ than that without added lactate. Doubling perfusion flow rate enhanced lactate uptake and VO ₂ by 120% and 40%, respectively. Glucose uptake was also increased (by 253%) with increased flow. Infusion of NA increased perfusion pressure, VO ₂ and glucose uptake similarly to those induced by increased flow rate. However, lactate uptake was inhibited by NA. This inhibition was not altered by the β-adrenergic antagonist propranolol. Vasopressin also showed similar effects to NA to decrease lactate uptake associated with increased VO ₂ and vasoconstriction. These data indicate that in the presence of a high arterial lactate concentration, NA has opposite effects from increased flow rate on skeletal muscle lactate uptake although both have similar effects on lactate release in the absence of arterial lactate. Inhibition of lactate uptake may relate to the vasoconstrictive action of NA.     

Hyperpolarized ketone body metabolism in the rat heart

The aim of this work was to investigate the use of ¹³C‐labelled acetoacetate and β‐hydroxybutyrate as novel hyperpolarized substrates in the study of cardiac metabolism. [1‐¹³C]Acetoacetate was synthesized by catalysed hydrolysis, and both it and [1‐¹³C]β‐hydroxybutyrate were hyperpolarized by dissolution dynamic nuclear polarization (DNP). Their metabolism was studied in isolated, perfused rat hearts. Hyperpolarized [1‐¹³C]acetoacetate metabolism was also studied in the in vivo rat heart in the fed and fasted states. Hyperpolarization of [1‐¹³C]acetoacetate and [1‐¹³C]β‐hydroxybutyrate provided liquid state polarizations of 8 ± 2% and 3 ± 1%, respectively. The hyperpolarized T₁ values for the two substrates were 28 ± 3 s (acetoacetate) and 20 ± 1 s (β‐hydroxybutyrate). Multiple downstream metabolites were observed within the perfused heart, including acetylcarnitine, citrate and glutamate. In the in vivo heart, an increase in acetylcarnitine production from acetoacetate was observed in the fed state, as well as a potential reduction in glutamate. In this work, methods for the generation of hyperpolarized [1‐¹³C]acetoacetate and [1‐¹³C]β‐hydroxybutyrate were investigated, and their metabolism was assessed in both isolated, perfused rat hearts and in the in vivo rat heart. These preliminary investigations show that DNP can be used as an effective in vivo probe of ketone body metabolism in the heart.     

Oxygen consumption in rabbit Langendorff hearts perfused with a saline medium

Isolated rabbit hearts were perfused according to Langendorff at a temperature of 38°C and a pressure of 5.9 kPa with gassed Tyrode solution. Gas mixtures containing 5 % CO₂ and 15, 20, 30, 60, or 95% O₂ in N₂ were used to saturate the perfusion medium. In some cases lactate (50 or 500 μM) was present in the medium perfusing the heart. Coronary flow (CF), oxygen pressure in the perfusion medium and in the cardiac effluent and lactate in the effluent were analysed in all experiments. The oxygen uptake in the hearts perfused with a medium equilibrated at atmospheric pressure with 95% O₂ and 5% CO₂ (oxygen pressure?87 kPa, oxygen content 19 ml × 1^-1), averaged 3 mix 100 g w.w. ^-1× min^-1. Reduction of the oxygen pressure in the perfusion medium resulted in an increase in CF and in the fractional extraction of oxygen from the medium, making it possible to maintain the heart's oxygen uptake (V?o₂) down to an oxygen pressure in the perfusion medium of about 24 kPa (oxygen content?5 ml × 1^-1, the perfusion medium equilibrated with 20% O₂ and 5%CO₂ in N₂). Myocardial lactate production was low during perfusion at pO₂? 87 kPa but increased rapidly when the oxygen pressure was lowered. The addition of lactate (500 /°M) to the perfusion medium at pO₂? 87 kP induced a fractional uptake of about 20%. It is concluded that the V?o₂ observed during perfusion at pO₂? 87 kPa mainly reflects aerobic myocardial metabolism in this preparation. This assumption is based on the facts that coronary flow and fractional oxygen extraction are submaximal and that a considerable uptake of lactate occurs concurrently with a very limited production. However, even moderate reduction of the oxygen pressure in the perfusion medium (to ? 61 kPa) is followed by a significant increase in lactate production, indicating that myocardial oxygenation is inefficient.     

In Vitro Work Load and Rat Heart Metabolism

The isolated working rat heart preparation was used to study the effects of well defined work loads on heart protein synthesis. The rate of protein synthesis was evaluated by measuring the incorporation rate of phenylalanine-³H into whole heart protein. Increased pressure load (afterload) accelerated the protein synthesis. A stimulatory effect was demonstrated with glucose or palmitate as substrate and with 1 or 5 times the normal rat plasma levels of all amino acids in the perfusion medium. The protein synthesis of both control and overloaded hearts was significantly accelerated with palmitate as substrate or with high levels of all amino acids. The work load effect could, thus, be obtained under conditions optimal for heart protein synthesis in vitro. In contrast to the stimulatory effect of in-creased afterload the rate of protein synthesis was not changed when the left atrial filling pressure (preload) was increased, although the external heart work was raised several times. End systolic volume was markedly increased in pressure overloaded hearts compared to hearts perfused under control conditions or with increased preload. It is concluded that an increase in contractile tension during systole and/or an increase in the end systolic volume was a necessary requirement for the acceleration of protein synthesis.     

Relationship between ischaemic time and ischaemia/reperfusion injury in isolated Langendorff‐perfused mouse hearts

Myocardial functional recovery and creatine kinase (CK) release following various periods of ischaemia were investigated in isolated mouse hearts. The hearts were perfused in the Langendorff mode with pyruvate‐containing Krebs–Hensleit (KH) buffer under a constant perfusion pressure of 80 mmHg, and were subjected to either continuous perfusion or to 5, 15, 20, 25, 30, 45 or 60 min of global ischaemia followed by 45 min of reperfusion. In hearts subjected to ischaemic periods of 5, 15 or 20 min, there was a transient reduction in the left ventricular (LV) dP/dt max during the early phase of reperfusion, while the recovery at the end of reperfusion reached a level similar to that in hearts subjected to continuous perfusion. In hearts subjected to longer ischaemic periods, i.e. 25, 30, 45 or 60 min, the decrease in the cardiac performance was more pronounced and persistent, with significantly lower recovery in LV dP/dt max and higher LV end diastolic pressure (LVEDP) at the end of reperfusion than in the non‐ischaemic hearts. There were no significant differences in the recoveries in coronary flow or in heart rate (HR) between groups. Similarly to the functional recovery, the release of CK showed a clear ischaemic length‐related increase. In conclusion, the Langendorff‐perfused isolated mouse heart could be a valuable model for studies of myocardial ischaemia/reperfusion injury. Future studies using gene‐targeted mice would add valuable knowledge to the understanding of myocardial ischaemia/reperfusion injury.