Hyperpolarized 13C and 31P MRS detects differences in cardiac energetics, metabolism, and function in obesity, and responses following treatment.

Obesity is associated with important changes in cardiac energetics and function, and an increased risk of adverse cardiovascular outcomes. Multi-nuclear MRS and MRI techniques have the potential to provide a comprehensive non-invasive assessment of cardiac metabolic perturbation in obesity. A rat model of obesity was created by high-fat diet feeding. This model was characterized using in vivo hyperpolarized [1-13C]pyruvate and [2-13C]pyruvate MRS, echocardiography and perfused heart 31P MRS. Two groups of obese rats were subsequently treated with either caloric restriction or the glucagon-like peptide-1 analogue/agonist liraglutide, prior to reassessment. The model recapitulated cardiovascular consequences of human obesity, including mild left ventricular hypertrophy, and diastolic, but not systolic, dysfunction. Hyperpolarized 13C and 31P MRS demonstrated that obesity was associated with reduced myocardial pyruvate dehydrogenase flux, altered cardiac tricarboxylic acid (TCA) cycle metabolism, and impaired myocardial energetic status (lower phosphocreatine to adenosine triphosphate ratio and impaired cardiac ΔG~ATP). Both caloric restriction and liraglutide treatment were associated with normalization of metabolic changes, alongside improvement in cardiac diastolic function. In this model of obesity, hyperpolarized 13C and 31P MRS demonstrated abnormalities in cardiac metabolism at multiple levels, including myocardial substrate selection, TCA cycle, and high-energy phosphorus metabolism. Metabolic changes were linked with impairment of diastolic function and were reversed in concert following either caloric restriction or liraglutide treatment. With hyperpolarized 13C and 31P techniques now available for human use, the findings support a role for multi-nuclear MRS in the development of new therapies for obesity.

Noninvasive In Vivo Assessment of Cardiac Metabolism in the Healthy and Diabetic Human Heart Using Hyperpolarized 13C MRI.

RATIONALE: The recent development of hyperpolarized 13C magnetic resonance spectroscopy has made it possible to measure cellular metabolism in vivo, in real time. OBJECTIVE: By comparing participants with and without type 2 diabetes mellitus (T2DM), we report the first case-control study to use this technique to record changes in cardiac metabolism in the healthy and diseased human heart. METHODS AND RESULTS: Thirteen people with T2DM (glycated hemoglobin, 6.9±1.0%) and 12 age-matched healthy controls underwent assessment of cardiac systolic and diastolic function, myocardial energetics (31P-magnetic resonance spectroscopy), and lipid content (1H-magnetic resonance spectroscopy) in the fasted state. In a subset (5 T2DM, 5 control), hyperpolarized [1-13C]pyruvate magnetic resonance spectra were also acquired and in 5 of these participants (3 T2DM, 2 controls), this was successfully repeated 45 minutes after a 75 g oral glucose challenge. Downstream metabolism of [1-13C]pyruvate via PDH (pyruvate dehydrogenase, [13C]bicarbonate), lactate dehydrogenase ([1-13C]lactate), and alanine transaminase ([1-13C]alanine) was assessed. Metabolic flux through cardiac PDH was significantly reduced in the people with T2DM (Fasted: 0.0084±0.0067 [Control] versus 0.0016±0.0014 [T2DM], Fed: 0.0184±0.0109 versus 0.0053±0.0041; P=0.013). In addition, a significant increase in metabolic flux through PDH was observed after the oral glucose challenge (P<0.001). As is characteristic of diabetes mellitus, impaired myocardial energetics, myocardial lipid content, and diastolic function were also demonstrated in the wider study cohort. CONCLUSIONS: This work represents the first demonstration of the ability of hyperpolarized 13C magnetic resonance spectroscopy to noninvasively assess physiological and pathological changes in cardiac metabolism in the human heart. In doing so, we highlight the potential of the technique to detect and quantify metabolic alterations in the setting of cardiovascular disease.

Assessing the optimal preparation strategy to minimize the variability of cardiac pyruvate dehydrogenase flux measurements with hyperpolarized MRS.

Hyperpolarized [1-13 C] pyruvate MRS can measure cardiac pyruvate dehydrogenase (PDH) flux in vivo through 13 C-label incorporation into bicarbonate. Using this technology, substrate availability as well as pathology have been shown to modulate PDH flux. Clinical protocols attempt to standardize PDH flux with oral glucose loading prior to scanning, while rodents in preclinical studies are usually scanned in the fed state. We aimed to establish which strategy was optimal to maximize PDH flux and minimize its variability in both control and Type II diabetic rats, without affecting the pathological variation being assessed. We found similar variances in the bicarbonate to pyruvate ratio, reflecting PDH flux, in fed and fasted/glucose-loaded animals, which showed no statistically significant differences. Furthermore, fasting/glucose loading did not alter the low PDH flux seen in Type II diabetic rats. Overall this suggests that preclinical cardiac hyperpolarized magnetic resonance studies could be performed either in the fed or in the fasted/glucose-loaded state. Centres planning to start new clinical studies with cardiac hyperpolarized magnetic resonance in man may find it beneficial to run small proof-of-concept trials to determine whether metabolic standardizations by oral or intravenous glucose load are beneficial compared with scanning patients in the fed state.

The 'Goldilocks zone' of fatty acid metabolism; to ensure that the relationship with cardiac function is just right.

Fatty acids (FA) are the main fuel used by the healthy heart to power contraction, supplying 60-70% of the ATP required. FA generate more ATP per carbon molecule than glucose, but require more oxygen to produce the ATP, making them a more energy dense but less oxygen efficient fuel compared with glucose. The pathways involved in myocardial FA metabolism are regulated at various subcellular levels, and can be divided into sarcolemmal FA uptake, cytosolic activation and storage, mitochondrial uptake and ß-oxidation. An understanding of the critical involvement of each of these steps has been amassed from genetic mouse models, where forcing the heart to metabolize too much or too little fat was accompanied by cardiac contractile dysfunction and hypertrophy. In cardiac pathologies, such as heart disease and diabetes, aberrations in FA metabolism occur concomitantly with changes in cardiac function. In heart failure, FA oxidation is decreased, correlating with systolic dysfunction and hypertrophy. In contrast, in type 2 diabetes, FA oxidation and triglyceride storage are increased, and correlate with diastolic dysfunction and insulin resistance. Therefore, too much FA metabolism is as detrimental as too little FA metabolism in these settings. Therapeutic compounds that rebalance FA metabolism may provide a mechanism to improve cardiac function in disease. Just like Goldilocks and her porridge, the heart needs to maintain FA metabolism in a zone that is 'just right' to support contractile function.

Simultaneous in vivo assessment of cardiac and hepatic metabolism in the diabetic rat using hyperpolarized MRS.

Understanding and assessing diabetic metabolism is vital for monitoring disease progression and improving treatment of patients. In vivo assessments, using MRI and MRS, provide non-invasive and accurate measurements, and the development of hyperpolarized 13 C spectroscopy in particular has been demonstrated to provide valuable metabolic data in real time. Until now, studies have focussed on individual organs. However, diabetes is a systemic disease affecting multiple tissues in the body. Therefore, we have developed a technique to simultaneously measure metabolism in both the heart and liver during a single acquisition. A hyperpolarized 13 C MRS protocol was developed to allow acquisition of metabolic data from the heart and liver during a single scan. This protocol was subsequently used to assess metabolism in the heart and liver of seven control male Wistar rats and seven diabetic rats (diabetes was induced by three weeks of high-fat feeding and a 30 mg/kg injection of streptozotocin). Using our new acquisition, we observed decreased cardiac and hepatic pyruvate dehydrogenase flux in our diabetic rat model. These diabetic rats also had increased blood glucose levels, decreased insulin, and increased hepatic triglycerides. Decreased production of hepatic [1-13 C]alanine was observed in the diabetic group, but this change was not present in the hearts of the same diabetic animals. We have demonstrated the ability to measure cardiac and hepatic metabolism simultaneously, with sufficient sensitivity to detect metabolic alterations in both organs. Further, we have non-invasively observed the different reactions of the heart and liver to the metabolic challenge of diabetes.

Hyperpolarized butyrate: a metabolic probe of short chain fatty acid metabolism in the heart.

PURPOSE: Butyrate, a short chain fatty acid, was studied as a novel hyperpolarized substrate for use in dynamic nuclear polarization enhanced magnetic resonance spectroscopy experiments, to define the pathways of short chain fatty acid and ketone body metabolism in real time. METHODS: Butyrate was polarized via the dynamic nuclear polarization process and subsequently dissolved to generate an injectable metabolic substrate. Metabolism was initially assessed in the isolated perfused rat heart, followed by evaluation in the in vivo rat heart. RESULTS: Hyperpolarized butyrate was generated with a polarization level of 7% and was shown to have a T1 relaxation time of 20 s. These physical characteristics were sufficient to enable assessment of multiple steps in its metabolism, with the ketone body acetoacetate and several tricarboxylic acid cycle intermediates observed both in vitro and in vivo. Metabolite to butyrate ratios of 0.1-0.4% and 0.5-2% were observed in vitro and in vivo respectively, similar to levels previously observed with hyperpolarized [2-(13) C]pyruvate. CONCLUSIONS: In this study, butyrate has been demonstrated to be a suitable hyperpolarized substrate capable of revealing multi-step metabolism in dynamic nuclear polarization experiments and providing information on the metabolism of fatty acids not currently achievable with other hyperpolarized substrates.

In vivo mouse cardiac hyperpolarized magnetic resonance spectroscopy.

BACKGROUND: Alterations in cardiac metabolism accompany many diseases of the heart. The advent of cardiac hyperpolarized magnetic resonance spectroscopy (MRS), via dynamic nuclear polarization (DNP), has enabled a greater understanding of the in vivo metabolic changes that occur as a consequence of myocardial infarction, hypertrophy and diabetes. However, all cardiac studies performed to date have focused on rats and larger animals, whereas more information could be gained through the study of transgenic mouse models of heart disease. Translation from the rat to the mouse is challenging, due in part to the reduced heart size (1/10(th)) and the increased heart rate (50%) in the mouse compared to the rat. METHODS AND RESULTS: In this study, we have investigated the in vivo metabolism of [1-(13)C]pyruvate in the mouse heart. To demonstrate the sensitivity of the method to detect alterations in pyruvate dehydrogenase (PDH) flux, two well characterised methods of PDH modulation were performed; overnight fasting and infusion of sodium dichloroacetate (DCA). Fasting resulted in an 85% reduction in PDH flux, whilst DCA infusion increased PDH flux by 123%. A comparison of three commonly used control mouse strains was performed revealing significant metabolic differences between strains. CONCLUSIONS: We have successfully demonstrated a hyperpolarized DNP protocol to investigate in vivo alterations within the diseased mouse heart. This technique offers a significant advantage over existing in vitro techniques as it reduces animal numbers and decreases biological variability. Thus [1-(13)C]pyruvate can be used to provide an in vivo cardiac metabolic profile of transgenic mice.

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.

Metabolic adaptation to chronic hypoxia in cardiac mitochondria.

Chronic hypoxia decreases cardiomyocyte respiration, yet the mitochondrial mechanisms remain largely unknown. We investigated the mitochondrial metabolic pathways and enzymes that were decreased following in vivo hypoxia, and questioned whether hypoxic adaptation was protective for the mitochondria. Wistar rats were housed in hypoxia (7 days acclimatisation and 14 days at 11% oxygen), while control rats were housed in normoxia. Chronic exposure to physiological hypoxia increased haematocrit and cardiac vascular endothelial growth factor, in the absence of weight loss and changes in cardiac mass. In both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria isolated from hypoxic hearts, state 3 respiration rates with fatty acid were decreased by 17-18%, and with pyruvate were decreased by 29-15%, respectively. State 3 respiration rates with electron transport chain (ETC) substrates were decreased only in hypoxic SSM, not in hypoxic IFM. SSM from hypoxic hearts had decreased activities of ETC complexes I, II and IV, which were associated with decreased reactive oxygen species generation and protection against mitochondrial permeability transition pore (MPTP) opening. In contrast, IFM from hypoxic hearts had decreased activity of the Krebs cycle enzyme, aconitase, which did not modify ROS production or MPTP opening. In conclusion, cardiac mitochondrial respiration was decreased following chronic hypoxia, associated with downregulation of different pathways in the two mitochondrial populations, determined by their subcellular location. Hypoxic adaptation was not deleterious for the mitochondria, in fact, SSM acquired increased protection against oxidative damage under the oxygen-limited conditions.

Haematological and biochemical pathology markers for a predictive model for ITU admission and death from COVID-19: A retrospective study.

Coronavirus disease (COVID-19) caused by SARS-CoV-2 has affected over 227 countries. Changes in haematological and biochemical characteristics in patients with COVID-19 are emerging as important features of the disease. This study aims to identify the pathological findings of COVID-19 patients at Bedford Hospital by analysing laboratory parameters that were identified as significant potential markers of COVID-19. Patients who were admitted to Bedford Hospital from March-July 2020 and had a positive swab for COVID were selected for this study. Clinical and laboratory data were collected using ICE system. Multiple haematological and biochemistry biomarkers were analysed using univariate and multivariate logistic regression to predict intensive therapy unit (ITU) admission and/or survival based on admission tests. Neutrophil-to-lymphocyte ratio (NLR) and C-reactive protein were elevated in most patients, irrespective of ITU status, representing a common outcome of COVID-19. This was driven by lymphopenia in 80% and neutrophilia in 42% of all patients. Multivariate logistic regression identified an increase in mortality associated with greater age, elevated NLR, alkaline phosphatase activity and hyperkalaemia. With the area under the receiver operating characteristic (ROC) curve of 0.706 +/- 0.04117, negative predictive value (NPV) 66.7% and positive predictive value (PPV) 64.9%. Analysis also revealed an association between increases in serum albumin and potassium concentrations and decreases in serum calcium, sodium and in prothrombin time, with admission to ITU. The area under the ROC curve of 0.8162 +/- 0.0403, NPV 63.3% and PPV 80.5%. These data suggest that using admission (within 4 days) measurements for haematological and biochemical markers, that we are able to predict outcome, whether that is survival or ITU admission.

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.

Metabolism.

Metabolism consists of a series of reactions that occur within cells of living organisms to sustain life. The process of metabolism involves many interconnected cellular pathways to ultimately provide cells with the energy required to carry out their function. The importance and the evolutionary advantage of these pathways can be seen as many remain unchanged by animals, plants, fungi, and bacteria. In eukaryotes, the metabolic pathways occur within the cytosol and mitochondria of cells with the utilisation of glucose or fatty acids providing the majority of cellular energy in animals. Metabolism is organised into distinct metabolic pathways to either maximise the capture of energy or minimise its use. Metabolism can be split into a series of chemical reactions that comprise both the synthesis and degradation of complex macromolecules known as anabolism or catabolism, respectively. The basic principles of energy consumption and production are discussed, alongside the biochemical pathways that make up fundamental metabolic processes for life.

Early detection of doxorubicin-induced cardiotoxicity in rats by its cardiac metabolic signature assessed with hyperpolarized MRI.

Doxorubicin (DOX) is a widely used chemotherapeutic agent that can cause serious cardiotoxic side effects culminating in congestive heart failure (HF). There are currently no clinical imaging techniques or biomarkers available to detect DOX-cardiotoxicity before functional decline. Mitochondrial dysfunction is thought to be a key factor driving functional decline, though real-time metabolic fluxes have never been assessed in DOX-cardiotoxicity. Hyperpolarized magnetic resonance imaging (MRI) can assess real-time metabolic fluxes in vivo. Here we show that cardiac functional decline in a clinically relevant rat-model of DOX-HF is preceded by a change in oxidative mitochondrial carbohydrate metabolism, measured by hyperpolarized MRI. The decreased metabolic fluxes were predominantly due to mitochondrial loss and additional mitochondrial dysfunction, and not, as widely assumed hitherto, to oxidative stress. Since hyperpolarized MRI has been successfully translated into clinical trials this opens up the potential to test cancer patients receiving DOX for early signs of cardiotoxicity.

CPT1a-Dependent Long-Chain Fatty Acid Oxidation Contributes to Maintaining Glucagon Secretion from Pancreatic Islets.

Glucagon, the principal hyperglycemic hormone, is secreted from pancreatic islet α cells as part of the counter-regulatory response to hypoglycemia. Hence, secretory output from α cells is under high demand in conditions of low glucose supply. Many tissues oxidize fat as an alternate energy substrate. Here, we show that glucagon secretion in low glucose conditions is maintained by fatty acid metabolism in both mouse and human islets, and that inhibiting this metabolic pathway profoundly decreases glucagon output by depolarizing α cell membrane potential and decreasing action potential amplitude. We demonstrate, by using experimental and computational approaches, that this is not mediated by the KATP channel, but instead due to reduced operation of the Na+-K+ pump. These data suggest that counter-regulatory secretion of glucagon is driven by fatty acid metabolism, and that the Na+-K+ pump is an important ATP-dependent regulator of α cell function.

Fatty Acids Prevent Hypoxia-Inducible Factor-1α Signaling Through Decreased Succinate in Diabetes.

Hypoxia-inducible factor (HIF)-1α is essential following a myocardial infarction (MI), and diabetic patients have poorer prognosis post-MI. Could HIF-1α activation be abnormal in the diabetic heart, and could metabolism be causing this? Diabetic hearts had decreased HIF-1α protein following ischemia, and insulin-resistant cardiomyocytes had decreased HIF-1α-mediated signaling and adaptation to hypoxia. This was due to elevated fatty acid (FA) metabolism preventing HIF-1α protein stabilization. FAs exerted their effect by decreasing succinate concentrations, a HIF-1α activator that inhibits the regulatory HIF hydroxylase enzymes. In vivo and in vitro pharmacological HIF hydroxylase inhibition restored HIF-1α accumulation and improved post-ischemic functional recovery in diabetes.

Nutritional Ketosis Alters Fuel Preference and Thereby Endurance Performance in Athletes.

Ketosis, the metabolic response to energy crisis, is a mechanism to sustain life by altering oxidative fuel selection. Often overlooked for its metabolic potential, ketosis is poorly understood outside of starvation or diabetic crisis. Thus, we studied the biochemical advantages of ketosis in humans using a ketone ester-based form of nutrition without the unwanted milieu of endogenous ketone body production by caloric or carbohydrate restriction. In five separate studies of 39 high-performance athletes, we show how this unique metabolic state improves physical endurance by altering fuel competition for oxidative respiration. Ketosis decreased muscle glycolysis and plasma lactate concentrations, while providing an alternative substrate for oxidative phosphorylation. Ketosis increased intramuscular triacylglycerol oxidation during exercise, even in the presence of normal muscle glycogen, co-ingested carbohydrate and elevated insulin. These findings may hold clues to greater human potential and a better understanding of fuel metabolism in health and disease.

Changes in the cardiac metabolome caused by perhexiline treatment in a mouse model of hypertrophic cardiomyopathy.

Energy depletion has been highlighted as an important contributor to the pathology of hypertrophic cardiomyopathy (HCM), a common inherited cardiac disease. Pharmacological reversal of energy depletion appears an attractive approach and the use of perhexiline has been proposed as it is thought to shift myocardial metabolism from fatty acid to glucose utilisation, increasing ATP production and myocardial efficiency. We used the Mybpc3-targeted knock-in mouse model of HCM to investigate changes in the cardiac metabolome following perhexiline treatment. Echocardiography indicated that perhexiline induced partial improvement of some, but not all hypertrophic parameters after six weeks. Non-targeted metabolomics, applying ultra-high performance liquid chromatography-mass spectrometry, described a phenotypic modification of the cardiac metabolome with 272 unique metabolites showing a statistically significant change (p < 0.05). Changes in fatty acids and acyl carnitines indicate altered fatty acid transport into mitochondria, implying reduction in fatty acid beta-oxidation. Increased glucose utilisation is indirectly implied through changes in the glycolytic, glycerol, pentose phosphate, tricarboxylic acid and pantothenate pathways. Depleted reduced glutathione and increased production of NADPH suggest reduction in oxidative stress. These data delineate the metabolic changes occurring during improvement of the HCM phenotype and indicate the requirements for further targeted interventions.

Impaired in vivo mitochondrial Krebs cycle activity after myocardial infarction assessed using hyperpolarized magnetic resonance spectroscopy.

BACKGROUND: Myocardial infarction (MI) is one of the leading causes of heart failure. An increasing body of evidence links alterations in cardiac metabolism and mitochondrial function with the progression of heart disease. The aim of this work was to, therefore, follow the in vivo mitochondrial metabolic alterations caused by MI, thereby allowing a greater understanding of the interplay between metabolic and functional abnormalities. METHODS AND RESULTS: Using hyperpolarized carbon-13 ((13)C)-magnetic resonance spectroscopy, in vivo alterations in mitochondrial metabolism were assessed for 22 weeks after surgically induced MI with reperfusion in female Wister rats. One week after MI, there were no detectable alterations in in vivo cardiac mitochondrial metabolism over the range of ejection fractions observed (from 28% to 84%). At 6 weeks after MI, in vivo mitochondrial Krebs cycle activity was impaired, with decreased (13)C-label flux into citrate, glutamate, and acetylcarnitine, which correlated with the degree of cardiac dysfunction. These changes were independent of alterations in pyruvate dehydrogenase flux. By 22 weeks, alterations were also seen in pyruvate dehydrogenase flux, which decreased at lower ejection fractions. These results were confirmed using in vitro analysis of enzyme activities and metabolomic profiles of key intermediates. CONCLUSIONS: The in vivo decrease in Krebs cycle activity in the 6-week post-MI heart may represent an early maladaptive phase in the metabolic alterations after MI in which reductions in Krebs cycle activity precede a reduction in pyruvate dehydrogenase flux. Changes in mitochondrial metabolism in heart disease are progressive and proportional to the degree of cardiac impairment.

Myocardial energy shortage and unmet anaplerotic needs in the fasted long-chain acyl-CoA dehydrogenase knockout mouse.

AIMS: The aim of this animal study is to assess fasting-induced changes in myocardial substrate metabolism and energy status as a consequence of mitochondrial long-chain fatty acid ß-oxidation deficiency, using magnetic resonance spectroscopy (MRS). METHODS AND RESULTS: Carbon-13 ((13)C) MRS of hyperpolarized [1-(13)C]pyruvate was used to assess in vivo pyruvate dehydrogenase (PDH) activity in fed and fasted wild-type (WT) mice and long-chain acyl-CoA dehydrogenase knockout (LCAD KO) mice. PDH activity decreased after fasting in both genotypes, but was 2.7-fold higher in fasted LCAD KO mice compared with fasted WT mice. Incorporation of the (13)C label into the myocardial malate and aspartate pools in fasted LCAD KO mice demonstrates enhanced activity of anaplerotic pathways in fasted LCAD KO hearts. These findings were corroborated by ex vivo assays revealing partially depleted pools of citric acid cycle intermediates in fasted LCAD KO myocardium, suggesting an increased, but unmet need for anaplerosis. The in vivo myocardial energy status, assessed using phosphorous-31 ((31)P) MRS, was lower in fasted LCAD KO mice than in fasted WT mice. CONCLUSION: This study revealed that the heart of fasted LCAD KO mice has an elevated reliance on glucose oxidation, in combination with an unmet demand for myocardial anaplerosis. Due to a lack of substrate availability, the sustained myocardial glucose uptake and PDH activity in LCAD KO mice are ineffective to maintain metabolic homeostasis during fasting, which is reflected by an impaired myocardial energy status in fasted LCAD KO mice.