Remodeling after myocardial infarction and effects of heart failure treatment investigated by hyperpolarized [1-13 C]pyruvate magnetic resonance spectroscopy.

PURPOSE: Hyperpolarized [1-13 C]pyruvate MRS can measure cardiac metabolism in vivo. We investigated whether [1-13 C]pyruvate MRS could predict left ventricular remodeling following myocardial infarction (MI), long-term left ventricular effects of heart failure medication, and could identify responders to treatment. METHODS: Thirty-five rats were scanned with hyperpolarized [1-13 C]pyruvate MRS 3 days after MI or sham surgery. The animals were re-examined after 30 days of therapy with ß-blockers and ACE-inhibitors (active group, n = 12), placebo treatment (placebo group, n = 13) or no treatment (sham group, n = 10). Furthermore, heart tissue mitochondrial respiratory capacity was assessed by high-resolution respirometry. Metabolic results were compared between groups, over time and correlated to functional MR data at each time point. RESULTS: At 30 ± 0.5 days post MI, left ventricular ejection fraction (LVEF) differed between groups (sham, 77% ± 1%; placebo, 52% ± 3%; active, 63% ± 2%, P < .001). Cardiac metabolism, measured by both hyperpolarized [1-13 C]pyruvate MRS and respirometry, neither differed between groups nor between baseline and follow-up. Three days post MI, low bicarbonate + CO2 /pyruvate ratio was associated with low LVEF. At follow-up, in the active group, a poor recovery of LVEF was associated with high bicarbonate + CO2 /pyruvate ratio, as measured by hyperpolarized MRS. CONCLUSION: In a rat model of moderate heart failure, medical treatment improved function, but did not on average influence [1-13 C]pyruvate flux as measured by MRS; however, responders to heart failure medication had reduced capacity for carbohydrate metabolism.

Hyperpolarized [1-13 C]pyruvate MRI can image the metabolic shift in cardiac metabolism between the fasted and fed state in a porcine model.

PURPOSE: Owing to its noninvasive nature, hyperpolarized MRI may improve delineation of myocardial metabolic derangement in heart disease. However, consistency may depend on the changeable nature of cardiac metabolism in relation to whole-body metabolic state. This study investigates the impact of feeding status on cardiac hyperpolarized MRI in a large animal model resembling human physiology. METHODS: Thirteen 30-kg pigs were subjected to an overnight fast, and 5 pigs were fed a carbohydrate-rich meal on the morning of the experiments. Vital parameters and blood samples were registered. All pigs were then scanned by hyperpolarized [1-13 C]pyruvate cardiac MRI, and results were compared between the 2 groups and correlated with circulating substrates and hormones. RESULTS: The fed group had higher blood glucose concentration and mean arterial pressure than the fasted group. Plasma concentrations of free fatty acids (FFAs) were decreased in the fed group, whereas plasma insulin concentrations were similar between groups. Hyperpolarized MRI showed that fed animals had increased lactate/pyruvate, alanine/pyruvate, and bicarbonate/pyruvate ratios. Metabolic ratios correlated negatively with FFA levels. CONCLUSION: Hyperpolarized MR can identify the effects of different metabolic states on cardiac metabolism in a large animal model. Unlike previous rodent studies, all metabolic derivatives of pyruvate increased in the myocardium of fed pigs. Carbohydrate-rich feeding seems to be a feasible model for standardized, large animal hyperpolarized MRI studies of myocardial carbohydrate metabolism.

Glucagon infusion alters the hyperpolarized 13 C-urea renal hemodynamic signature.

Renal urea handling is central to the urine concentrating mechanism, and as such the ability to image urea transport in the kidney is an important potential imaging biomarker for renal functional assessment. Glucagon levels associated with changes in dietary protein intake have been shown to influence renal urea handling; however, the exact mechanism has still to be fully understood. Here we investigate renal function and osmolite distribution using [13 C,15 N] urea dynamics and 23 Na distribution before and 60 min after glucagon infusion in six female rats. Glucagon infusion increased the renal [13 C,15 N] urea mean transit time by 14%, while no change was seen in the sodium distribution, glomerular filtration rate or oxygen consumption. This change is related to the well-known effect of increased urea excretion associated with glucagon infusion, independent of renal functional effects. This study demonstrates for the first time that hyperpolarized 13 C-urea enables monitoring of renal urinary excretion effects in vivo.

In Vivo Simulation of Magnesium Degradability Using a New Fluid Dynamic Bench Testing Approach.

The degradation rate of magnesium (Mg) alloys is a key parameter to develop Mg-based biomaterials and ensure in vivo-mechanical stability as well as to minimize hydrogen gas production, which otherwise can lead to adverse effects in clinical applications. However, in vitro and in vivo results of the same material often differ largely. In the present study, a dynamic test bench with several single bioreactor cells was constructed to measure the volume of hydrogen gas which evolves during magnesium degradation to indicate the degradation rate in vivo. Degradation medium comparable with human blood plasma was used to simulate body fluids. The media was pumped through the different bioreactor cells under a constant flow rate and 37 °C to simulate physiological conditions. A total of three different Mg groups were successively tested: Mg WE43, and two different WE43 plasma electrolytically oxidized (PEO) variants. The results were compared with other methods to detect magnesium degradation (pH, potentiodynamic polarization (PDP), cytocompatibility, SEM (scanning electron microscopy)). The non-ceramized specimens showed the highest degradation rates and vast standard deviations. In contrast, the two PEO samples demonstrated reduced degradation rates with diminished standard deviation. The pH values showed above-average constant levels between 7.4-7.7, likely due to the constant exchange of the fluids. SEM revealed severe cracks on the surface of WE43 after degradation, whereas the ceramized surfaces showed significantly decreased signs of corrosion. PDP results confirmed the improved corrosion resistance of both PEO samples. While WE43 showed slight toxicity in vitro, satisfactory cytocompatibility was achieved for the PEO test samples. In summary, the dynamic test bench constructed in this study enables reliable and simple measurement of Mg degradation to simulate the in vivo environment. Furthermore, PEO treatment of magnesium is a promising method to adjust magnesium degradation.

Acute renal metabolic effect of metformin assessed with hyperpolarised MRI in rats.

AIMS/HYPOTHESIS: Metformin inhibits hepatic mitochondrial glycerol phosphate dehydrogenase, thereby increasing cytosolic lactate and suppressing gluconeogenesis flux in the liver. This inhibition alters cytosolic and mitochondrial reduction-oxidation (redox) potential, which has been reported to protect organ function in several disease states including diabetes. In this study, we investigated the acute metabolic and functional changes induced by metformin in the kidneys of both healthy and insulinopenic Wistar rats used as a model of diabetes. METHODS: Diabetes was induced by intravenous injection of streptozotocin, and kidney metabolism in healthy and diabetic animals was investigated 4 weeks thereafter using hyperpolarised 13C-MRI, Clark-type electrodes and biochemical analysis. RESULTS: Metformin increased renal blood flow, but did not change total kidney oxygen consumption. In healthy rat kidneys, metformin increased [1-13C]lactate production and reduced mitochondrial [1-13C]pyruvate oxidation (decreased the 13C-bicarbonate/[1-13C]pyruvate ratio) within 30 min of administration. Corresponding alterations to indices of mitochondrial, cytosolic and whole-cell redox potential were observed. Pyruvate oxidation was maintained in the diabetic rats, suggesting that the diabetic state abrogates metabolic reprogramming caused by metformin. CONCLUSIONS/INTERPRETATION: This study demonstrates that metformin-induced acute metabolic alterations in healthy kidneys favoured anaerobic metabolism at the expense of aerobic metabolism. The results suggest that metformin directly alters the renal redox state, with elevated renal cytosolic redox states as well as decreased mitochondrial redox state. These findings suggest redox biology as a novel target to eliminate the renal complications associated with metformin treatment in individuals with impaired renal function.

Acute hypertensive stress imaged by cardiac hyperpolarized [1-13 C]pyruvate magnetic resonance.

PURPOSE: Deranged metabolism is now recognized as a key causal factor in a variety of heart diseases, and is being studied extensively. However, invasive methods may alter metabolism, and conventional imaging techniques measure tracer uptake but not downstream metabolism. These challenges may be overcome by hyperpolarized MR, a noninvasive technique currently crossing the threshold into human trials. The aim of this study was to image metabolic changes in the heart in response to endogastric glucose bolus and to acute hypertension. METHODS: Five postprandial pigs were scanned with hyperpolarized [1-13 C]pyruvate cardiac MR at baseline, after oral glucose bolus, and after infusion of angiotensin-II. RESULTS: No effect of glucose bolus was seen using hyperpolarized [1-13 C]pyruvate MR despite changes in circulating substrates. During angiotensin-II infusion, blood pressure increased 179% (P = 0.008) and ejection fraction decreased from 54 ± 2% to 47 ± 6% (P = 0.03) The hemodynamic changes were accompanied by increases in the hyperpolarized [1-13 C]pyruvate MR derived ratios of lactate/alanine (from 0.58 ± 0.13 to 0.78 ± 0.06, P = 0.03) and bicarbonate/alanine (from 0.55 ± 0.12 to 0.91 ± 0.14, P = 0.007). CONCLUSION: Glucose loading did not alter cardiac metabolism, but during acute hypertensive stress, cardiac aerobic, carbohydrate metabolism, and pyruvate-lactate exchange was altered. Hyperpolarized MR allows noninvasive evaluation of acute changes in cardiac metabolism. However, hemodynamics must be taken into account when interpreting the results.

Probing the cardiac malate-aspartate shuttle non-invasively using hyperpolarized [1,2-13 C2 ]pyruvate.

Previous studies have demonstrated that using hyperpolarized [2-13 C]pyruvate as a contrast agent can reveal 13 C signals from metabolites associated with the tricarboxylic acid (TCA) cycle. However, the metabolites detectable from TCA cycle-mediated oxidation of [2-13 C]pyruvate are the result of several metabolic steps. In the instance of the [5-13 C]glutamate signal, the amplitude can be modulated by changes to the rates of pyruvate dehydrogenase (PDH) flux, TCA cycle flux and metabolite pool size. Also key is the malate-aspartate shuttle, which facilitates the transport of cytosolic reducing equivalents into the mitochondria for oxidation via the malate-α-ketoglutarate transporter, a process coupled to the exchange of cytosolic malate for mitochondrial α-ketoglutarate. In this study, we investigated the mechanism driving the observed changes to hyperpolarized [2-13 C]pyruvate metabolism. Using hyperpolarized [1,2-13 C]pyruvate with magnetic resonance spectroscopy (MRS) in the porcine heart with different workloads, it was possible to probe 13 C-glutamate labeling relative to rates of cytosolic metabolism, PDH flux and TCA cycle turnover in a single experiment non-invasively. Via the [1-13 C]pyruvate label, we observed more than a five-fold increase in the cytosolic conversion of pyruvate to [1-13 C]lactate and [1-13 C]alanine with higher workload. 13 C-Bicarbonate production by PDH was increased by a factor of 2.2. Cardiac cine imaging measured a two-fold increase in cardiac output, which is known to couple to TCA cycle turnover. Via the [2-13 C]pyruvate label, we observed that 13 C-acetylcarnitine production increased 2.5-fold in proportion to the 13 C-bicarbonate signal, whereas the 13 C-glutamate metabolic flux remained constant on adrenergic activation. Thus, the 13 C-glutamate signal relative to the amount of 13 C-labeled acetyl-coenzyme A (acetyl-CoA) entering the TCA cycle was decreased by 40%. The data strongly suggest that NADH (reduced form of nicotinamide adenine dinucleotide) shuttling from the cytosol to the mitochondria via the malate-aspartate shuttle is limited on adrenergic activation. Changes in [5-13 C]glutamate production from [2-13 C]pyruvate may play an important future role in non-invasive myocardial assessment in patients with cardiovascular diseases, but careful interpretation of the results is required.

Desmopressin use in pediatric nocturnal enuresis patients: is there a sex difference in prescription patterns?

Desmopressin is a long-established treatment for nocturnal enuresis with clear guidelines regarding its usage. A sex difference in renal sensitivity has recently been reported in adults. The objective of this study was to investigate real-life desmopressin prescription in the Danish pediatric population, and prescription patterns which may reflect a sex difference in pediatric usage. Formulation, dose, treatment duration, and safety (hyponatremia) were investigated. 40,596 children received 214,220 desmopressin prescriptions between 2004 and 2011 in the Danish National Prescription Registry. Data were linked to hyponatremia diagnoses from the National Patient Registry. Although the lowest recommended dose of desmopressin oral lyophilisate is 120 µg, around a fifth of children were prescribed 60 µg for long-term use. A greater proportion of girls (22.6%) than boys (19.8%) received this low dose. Treatment duration was longer for boys than girls on oral lyophilisate (mean 489-524 vs. 414-462 days) and tablet (0.1 mg: 204 vs. 161 days). Prescribed daily dose was consistent with time between prescriptions, indicating no significant drug holidays. There were no admissions for hyponatremia during the observation period. CONCLUSION: Danish national prescription data on pediatric desmopressin dosage are consistent with a greater sensitivity to desmopressin in girls than boys. Further studies are required. What is Known: • Desmopressin has been used for pediatric nocturnal enuresis for decades • Recent evidence suggests a sex difference in desmopressin sensitivity in adults What is New: • For the first time, desmopressin prescription practices in nocturnal enuresis are documented for an entire country • A higher proportion of girls than boys received a low dose of desmopressin, consistent with the sex difference in sensitivity reported in adults.

Determination of the renal concentration capacity following intravenous administration of dDAVP in healthy humans.

The synthetic AVP analogue 1-desamino-8-d-arginine-vasopressin (dDAVP) is used for treatment of polyuric disorders. Lack of commercially available assays limits the usefulness of dDAVP as a diagnostic tool in the assessment of renal concentrating capacity. We aimed to develop a specific radioimmunoassay (RIA) for determination of plasma dDAVP (pdDAVP) in order to investigate the relationship between pdDAVP levels and urine osmolality (Uosm). Further, we aimed to determine the onset, duration, and maximum concentrating capacity following intravenous (i.v.) bolus dDAVP injection. The dDAVP assay was based on a well-established RIA for measurements of AVP. Fourteen healthy subjects (aged 15-18 years) participated. Blood and urine samples were collected prior to and after i.v. bolus of 0.03 µg/kg dDAVP. Diuresis and Uosm was measured for nine hours following dDAVP administration. PdDAVP and Uosm were analyzed.We established a specific RIA for the measurement of pdDAVP. All subjects reached maximal pdDAVP concentration (Cmax) 30 minutes following infusion, and a rise in Uosm after 60 minutes. Maximal Uosm varied between subjects, with no direct correlation to the achieved pdDAVP levels. We found no significant intra-individual variation between two dDAVP infusions and the effect was reproducible in terms of Cmax and maximal Uosm. We characterized the relationship between pdDAVP and Uosm after dDAVP bolus injection in healthy adolescents using our dDAVP assay. Maximal Uosm achieved correlated with the baseline Uosm levels and seemed unrelated to achieved pdDAVP levels. The urine concentrating response was maintained at least eight hours.

Novel ketone diet enhances physical and cognitive performance.

Ketone bodies are the most energy-efficient fuel and yield more ATP per mole of substrate than pyruvate and increase the free energy released from ATP hydrolysis. Elevation of circulating ketones via high-fat, low-carbohydrate diets has been used for the treatment of drug-refractory epilepsy and for neurodegenerative diseases, such as Parkinson's disease. Ketones may also be beneficial for muscle and brain in times of stress, such as endurance exercise. The challenge has been to raise circulating ketone levels by using a palatable diet without altering lipid levels. We found that blood ketone levels can be increased and cholesterol and triglycerides decreased by feeding rats a novel ketone ester diet: chow that is supplemented with (R)-3-hydroxybutyl (R)-3-hydroxybutyrate as 30% of calories. For 5 d, rats on the ketone diet ran 32% further on a treadmill than did control rats that ate an isocaloric diet that was supplemented with either corn starch or palm oil (P < 0.05). Ketone-fed rats completed an 8-arm radial maze test 38% faster than did those on the other diets, making more correct decisions before making a mistake (P < 0.05). Isolated, perfused hearts from rats that were fed the ketone diet had greater free energy available from ATP hydrolysis during increased work than did hearts from rats on the other diets as shown by using [31P]-NMR spectroscopy. The novel ketone diet, therefore, improved physical performance and cognitive function in rats, and its energy-sparing properties suggest that it may help to treat a range of human conditions with metabolic abnormalities.-Murray, A. J., Knight, N. S., Cole, M. A., Cochlin, L. E., Carter, E., Tchabanenko, K., Pichulik, T., Gulston, M. K., Atherton, H. J., Schroeder, M. A., Deacon, R. M. J., Kashiwaya, Y., King, M. T., Pawlosky, R., Rawlins, J. N. P., Tyler, D. J., Griffin, J. L., Robertson, J., Veech, R. L., Clarke, K. Novel ketone diet enhances physical and cognitive performance.

Extramitochondrial domain rich in carbonic anhydrase activity improves myocardial energetics.

CO2 is produced abundantly by cardiac mitochondria. Thus an efficient means for its venting is required to support metabolism. Carbonic anhydrase (CA) enzymes, expressed at various sites in ventricular myocytes, may affect mitochondrial CO2 clearance by catalyzing CO2 hydration (to H(+) and HCO3(-)), thereby changing the gradient for CO2 venting. Using fluorescent dyes to measure changes in pH arising from the intracellular hydration of extracellularly supplied CO2, overall CA activity in the cytoplasm of isolated ventricular myocytes was found to be modest (2.7-fold above spontaneous kinetics). Experiments on ventricular mitochondria demonstrated negligible intramitochondrial CA activity. CA activity was also investigated in intact hearts by (13)C magnetic resonance spectroscopy from the rate of H(13)CO3(-) production from (13)CO2 released specifically from mitochondria by pyruvate dehydrogenase-mediated metabolism of hyperpolarized [1-(13)C]pyruvate. CA activity measured upon [1-(13)C]pyruvate infusion was fourfold higher than the cytoplasm-averaged value. A fluorescent CA ligand colocalized with a mitochondrial marker, indicating that mitochondria are near a CA-rich domain. Based on immunoreactivity, this domain comprises the nominally cytoplasmic CA isoform CAII and sarcoplasmic reticulum-associated CAXIV. Inhibition of extramitochondrial CA activity acidified the matrix (as determined by fluorescence measurements in permeabilized myocytes and isolated mitochondria), impaired cardiac energetics (indexed by the phosphocreatine-to-ATP ratio measured by (31)P magnetic resonance spectroscopy of perfused hearts), and reduced contractility (as measured from the pressure developed in perfused hearts). These data provide evidence for a functional domain of high CA activity around mitochondria to support CO2 venting, particularly during elevated and fluctuating respiratory activity. Aberrant distribution of CA activity therefore may reduce the heart's energetic efficiency.

Multicentric carpotarsal osteolysis is caused by mutations clustering in the amino-terminal transcriptional activation domain of MAFB.

Multicentric carpotarsal osteolysis (MCTO) is a rare skeletal dysplasia characterized by aggressive osteolysis, particularly affecting the carpal and tarsal bones, and is frequently associated with progressive renal failure. Using exome capture and next-generation sequencing in five unrelated simplex cases of MCTO, we identified previously unreported missense mutations clustering within a 51 base pair region of the single exon of MAFB, validated by Sanger sequencing. A further six unrelated simplex cases with MCTO were also heterozygous for previously unreported mutations within this same region, as were affected members of two families with autosomal-dominant MCTO. MAFB encodes a transcription factor that negatively regulates RANKL-induced osteoclastogenesis and is essential for normal renal development. Identification of this gene paves the way for development of novel therapeutic approaches for this crippling disease and provides insight into normal bone and kidney development.

Desmopressin as an adjuvant to opioids or NSAIDs in treatment of renal colic: a nationwide register-based study.

AIMS: Desmopressin has been reported to be effective as an adjuvant to opioids or NSAIDs in management of pain in renal colic; however real-life data are lacking on the utilisation of desmopressin in this patient segment. METHODS: The Danish National Prescription Registry data-linked with Danish National Patient Registry during a 3-year period from 2009 to 2011 was used to study prescriptions for desmopressin in renal colic. RESULTS: We identified 888 desmopressin prescriptions for renal colic, dispensed to 95 patients. The mean treatment period was 159 days, with a large variation up to a maximum of 924 days. Approximately two thirds of patients received dosing instructions to administer the drug 4 times daily to provide 24-h antidiuretic coverage. Among concomitant opioids and NSAIDs, tramadol and ibuprofen were prescribed most frequently. Antidepressants and diuretics were also widely used. A clear sex difference was seen, with female renal colic patients having three times more prescriptions overall than males, and in particular receiving more antidepressants and psychotropic drugs. A total of 4 (4.2%) of the patients experienced hospital admissions because of hyponatraemia or polydipsia during the 3-year period. We confirmed a previous case report that nephrolithiasis could be at least an occasional complication of successful therapy of Central Diabetes Insipidus (CDI) with desmopressin, identifying 12 CDI patients in total, or 2.4% of all Danish CDI patients in that period, who were also treated for renal colic. CONCLUSION: In summary, these real-life prescription data provide exact epidemiological measures on desmopressin utilisation in renal colic.

The Cardioprotective Effects of Semaglutide Exceed Those of Dietary Weight Loss in Mice With HFpEF.

Obesity-related heart failure with preserved ejection fraction (HFpEF) has become a well-recognized HFpEF subphenotype. Targeting the unfavorable cardiometabolic profile may represent a rational treatment strategy. This study investigated semaglutide, a glucagon-like peptide-1 receptor agonist that induces significant weight loss in patients with obesity and/or type 2 diabetes mellitus and has been associated with improved cardiovascular outcomes. In a mouse model of HFpEF that was caused by advanced aging, female sex, obesity, and type 2 diabetes mellitus, semaglutide, compared with weight loss induced by pair feeding, improved the cardiometabolic profile, cardiac structure, and cardiac function. Mechanistically, transcriptomic, and proteomic analyses revealed that semaglutide improved left ventricular cytoskeleton function and endothelial function and restores protective immune responses in visceral adipose tissue. Strikingly, treatment with semaglutide induced a wide array of favorable cardiometabolic effects beyond the effect of weight loss by pair feeding. Glucagon-like peptide-1 receptor agonists may therefore represent an important novel therapeutic option for treatment of HFpEF, especially when obesity-related.

Role of pyruvate dehydrogenase inhibition in the development of hypertrophy in the hyperthyroid rat heart: a combined magnetic resonance imaging and hyperpolarized magnetic resonance spectroscopy study.

BACKGROUND: Hyperthyroidism increases heart rate, contractility, cardiac output, and metabolic rate. It is also accompanied by alterations in the regulation of cardiac substrate use. Specifically, hyperthyroidism increases the ex vivo activity of pyruvate dehydrogenase kinase, thereby inhibiting glucose oxidation via pyruvate dehydrogenase. Cardiac hypertrophy is another effect of hyperthyroidism, with an increase in the abundance of mitochondria. Although the hypertrophy is initially beneficial, it can eventually lead to heart failure. The aim of this study was to use hyperpolarized magnetic resonance spectroscopy to investigate the rate and regulation of in vivo pyruvate dehydrogenase flux in the hyperthyroid heart and to establish whether modulation of flux through pyruvate dehydrogenase would alter cardiac hypertrophy. METHODS AND RESULTS: Hyperthyroidism was induced in 18 male Wistar rats with 7 daily intraperitoneal injections of freshly prepared triiodothyronine (0.2 mg x kg(-1) x d(-1)). In vivo pyruvate dehydrogenase flux, assessed with hyperpolarized magnetic resonance spectroscopy, was reduced by 59% in hyperthyroid animals (0.0022 ± 0.0002 versus 0.0055 ± 0.0005 second(-1); P=0.0003), and this reduction was completely reversed by both short- and long-term delivery of dichloroacetic acid, a pyruvate dehydrogenase kinase inhibitor. Hyperpolarized [2-(13)C]pyruvate was also used to evaluate Krebs cycle metabolism and demonstrated a unique marker of anaplerosis, the level of which was significantly increased in the hyperthyroid heart. Cine magnetic resonance imaging showed that long-term dichloroacetic acid treatment significantly reduced the hypertrophy observed in hyperthyroid animals (100 ± 20 versus 200 ± 30 mg; P=0.04) despite no change in the increase observed in cardiac output. CONCLUSIONS: This work has demonstrated that inhibition of glucose oxidation in the hyperthyroid heart in vivo is mediated by pyruvate dehydrogenase kinase. Relieving this inhibition can increase the metabolic flexibility of the hyperthyroid heart and reduce the level of hypertrophy that develops while maintaining the increased cardiac output required to meet the higher systemic metabolic demand.

Simultaneous investigation of cardiac pyruvate dehydrogenase flux, Krebs cycle metabolism and pH, using hyperpolarized [1,2-(13)C2]pyruvate in vivo.

(13)C MR spectroscopy studies performed on hearts ex vivo and in vivo following perfusion of prepolarized [1-(13)C]pyruvate have shown that changes in pyruvate dehydrogenase (PDH) flux may be monitored non-invasively. However, to allow investigation of Krebs cycle metabolism, the (13)C label must be placed on the C2 position of pyruvate. Thus, the utilization of either C1 or C2 labeled prepolarized pyruvate as a tracer can only afford a partial view of cardiac pyruvate metabolism in health and disease. If the prepolarized pyruvate molecules were labeled at both C1 and C2 positions, then it would be possible to observe the downstream metabolites that were the results of both PDH flux ((13)CO(2) and H(13)CO(3)(-)) and Krebs cycle flux ([5-(13)C]glutamate) with a single dose of the agent. Cardiac pH could also be monitored in the same experiment, but adequate SNR of the (13)CO(2) resonance may be difficult to obtain in vivo. Using an interleaved selective RF pulse acquisition scheme to improve (13)CO(2) detection, the feasibility of using dual-labeled hyperpolarized [1,2-(13)C(2)]pyruvate as a substrate for dynamic cardiac metabolic MRS studies to allow simultaneous investigation of PDH flux, Krebs cycle flux and pH, was demonstrated in vivo.

Determining the in vivo regulation of cardiac pyruvate dehydrogenase based on label flux from hyperpolarised [1-13C]pyruvate.

Pyruvate dehydrogenase (PDH) is a key regulator of cardiac substrate selection and is regulated by both pyruvate dehydrogenase kinase (PDK)-mediated phosphorylation and feedback inhibition. The extent to which chronic upregulation of PDK protein levels, acutely increased PDK activity and acute feedback inhibition limit PDH flux remains unclear because existing in vitro assessment methods inherently disrupt the regulation of the enzyme complex. We have demonstrated previously that hyperpolarised (13)C-labelled metabolic tracers coupled with MRS can monitor flux through PDH in vivo. The aim of this study was to determine the relative contributions of acute and chronic changes in PDK and PDH activities to in vivo myocardial PDH flux. We examined both fed and fasted rats with either hyperpolarised [1-(13)C]pyruvate alone or hyperpolarised [1-(13)C]pyruvate co-infused with malate [to modulate mitochondrial nicotinamide adenine dinucleotide (NADH/NAD(+)) and acetyl-coenzyme A (acetyl-CoA)/CoA ratios, which alter both PDH activity and flux]. To confirm the metabolic fate of infused malate, we performed in vitro (1)H NMR spectroscopy on cardiac tissue extracts. We observed that, in fed rats, where PDH activity was high, the presence of malate increased PDH flux by 27%, whereas, in the fasted state, malate infusion had no effect on PDH flux. These observations suggest that pyruvate oxidation is limited by feedback inhibition from acetyl-CoA only when PDH activity is high. Therefore, in the case of PDH, and potentially other enzymes, hyperpolarised (13)C MRI can be used to assess noninvasively enzymatic regulation.

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-(13)C]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 ß-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.

In vivo assessment of pyruvate dehydrogenase flux in the heart using hyperpolarized carbon-13 magnetic resonance.

The advent of hyperpolarized (13)C magnetic resonance (MR) has provided new potential for the real-time visualization of in vivo metabolic processes. The aim of this work was to use hyperpolarized [1-(13)C]pyruvate as a metabolic tracer to assess noninvasively the flux through the mitochondrial enzyme complex pyruvate dehydrogenase (PDH) in the rat heart, by measuring the production of bicarbonate (H(13)CO(3)(-)), a byproduct of the PDH-catalyzed conversion of [1-(13)C]pyruvate to acetyl-CoA. By noninvasively observing a 74% decrease in H(13)CO(3)(-) production in fasted rats compared with fed controls, we have demonstrated that hyperpolarized (13)C MR is sensitive to physiological perturbations in PDH flux. Further, we evaluated the ability of the hyperpolarized (13)C MR technique to monitor disease progression by examining PDH flux before and 5 days after streptozotocin induction of type 1 diabetes. We detected decreased H(13)CO(3)(-) production with the onset of diabetes that correlated with disease severity. These observations were supported by in vitro investigations of PDH activity as reported in the literature and provided evidence that flux through the PDH enzyme complex can be monitored noninvasively, in vivo, by using hyperpolarized (13)C MR.

Real-time assessment of Krebs cycle metabolism using hyperpolarized 13C magnetic resonance spectroscopy.

The Krebs cycle plays a fundamental role in cardiac energy production and is often implicated in the energetic imbalance characteristic of heart disease. In this study, we measured Krebs cycle flux in real time in perfused rat hearts using hyperpolarized magnetic resonance spectroscopy (MRS). [2-(13)C]Pyruvate was hyperpolarized and infused into isolated perfused hearts in both healthy and postischemic metabolic states. We followed the enzymatic conversion of pyruvate to lactate, acetylcarnitine, citrate, and glutamate with 1 s temporal resolution. The appearance of (13)C-labeled glutamate was delayed compared with that of other metabolites, indicating that Krebs cycle flux can be measured directly. The production of (13)C-labeled citrate and glutamate was decreased postischemia, as opposed to lactate, which was significantly elevated. These results showed that the control and fluxes of the Krebs cycle in heart disease can be studied using hyperpolarized [2-(13)C]pyruvate.