Assessment of Cardiac Energy Metabolism, Function, and Physiology in Patients With Heart Failure Taking Empagliflozin: The Randomized, Controlled EMPA-VISION Trial.

BACKGROUND: Sodium-glucose co-transporter 2 inhibitors (SGLT2i) have emerged as a paramount treatment for patients with heart failure (HF), irrespective of underlying reduced or preserved ejection fraction. However, a definite cardiac mechanism of action remains elusive. Derangements in myocardial energy metabolism are detectable in all HF phenotypes, and it was proposed that SGLT2i may improve energy production. The authors aimed to investigate whether treatment with empagliflozin leads to changes in myocardial energetics, serum metabolomics, and cardiorespiratory fitness. METHODS: EMPA-VISION (Assessment of Cardiac Energy Metabolism, Function and Physiology in Patients With Heart Failure Taking Empagliflozin) is a prospective, randomized, double-blind, placebo-controlled, mechanistic trial that enrolled 72 symptomatic patients with chronic HF with reduced ejection fraction (HFrEF; n=36; left ventricular ejection fraction ≤40%; New York Heart Association class ≥II; NT-proBNP [N-terminal pro-B-type natriuretic peptide] ≥125 pg/mL) and HF with preserved ejection fraction (HFpEF; n=36; left ventricular ejection fraction ≥50%; New York Heart Association class ≥II; NT-proBNP ≥125 pg/mL). Patients were stratified into respective cohorts (HFrEF versus HFpEF) and randomly assigned to empagliflozin (10 mg; n=35: 17 HFrEF and 18 HFpEF) or placebo (n=37: 19 HFrEF and 18 HFpEF) once daily for 12 weeks. The primary end point was a change in the cardiac phosphocreatine:ATP ratio (PCr/ATP) from baseline to week 12, determined by phosphorus magnetic resonance spectroscopy at rest and during peak dobutamine stress (65% of age-maximum heart rate). Mass spectrometry on a targeted set of 19 metabolites was performed at baseline and after treatment. Other exploratory end points were investigated. RESULTS: Empagliflozin treatment did not change cardiac energetics (ie, PCr/ATP) at rest in HFrEF (adjusted mean treatment difference [empagliflozin - placebo], -0.25 [95% CI, -0.58 to 0.09]; P=0.14) or HFpEF (adjusted mean treatment difference, -0.16 [95% CI, -0.60 to 0.29]; P=0.47]. Likewise, there were no changes in PCr/ATP during dobutamine stress in HFrEF (adjusted mean treatment difference, -0.13 [95% CI, -0.35 to 0.09]; P=0.23) or HFpEF (adjusted mean treatment difference, -0.22 [95% CI, -0.66 to 0.23]; P=0.32). No changes in serum metabolomics or levels of circulating ketone bodies were observed. CONCLUSIONS: In patients with either HFrEF or HFpEF, treatment with 10 mg of empagliflozin once daily for 12 weeks did not improve cardiac energetics or change circulating serum metabolites associated with energy metabolism when compared with placebo. Based on our results, it is unlikely that enhancing cardiac energy metabolism mediates the beneficial effects of SGLT2i in HF. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03332212.

Compartment-based reconstruction of 3D acquisition-weighted 31 P cardiac magnetic resonance spectroscopic imaging at 7 T: A reproducibility study.

Even at 7 T, cardiac 31 P magnetic resonance spectroscopic imaging (MRSI) is fundamentally limited by low signal-to-noise ratio (SNR), leading to long scan times and poor temporal and spatial resolutions. Compartment-based reconstruction algorithms such as magnetic resonance spectroscopy with linear algebraic modeling (SLAM) and spectral localization by imaging (SLIM) may improve SNR or reduce scan time without changes to acquisition. Here, we compare the repeatability and SNR performance of these compartment-based methods, applied to three different acquisition schemes at 7 T. Twelve healthy volunteers were scanned twice. Each scan session consisted of a 6.5-min 3D acquisition-weighted (AW) cardiac 31 P phase encode-based MRSI acquisition and two 6.5-min truncated k-space acquisitions with increased averaging (4 × 4 × 4 central k-space phase encodes and fractional SLAM [fSLAM] optimized k-space phase encodes). Spectra were reconstructed using (i) AW Fourier reconstruction; (ii) AW SLAM; (iii) AW SLIM; (iv) 4 × 4 × 4 SLAM; (v) 4 × 4 × 4 SLIM; and (vi) fSLAM acquisition-reconstruction combinations. The phosphocreatine-to-adenosine triphosphate (PCr/ATP) ratio, the PCr SNR, and spatial response functions were computed, in addition to coefficients of reproducibility and variability. Using the compartment-based reconstruction algorithms with the AW 31 P acquisition resulted in a significant increase in SNR compared with previously published Fourier-based MRSI reconstruction methods while maintaining the measured PCr/ATP ratio and improving interscan reproducibility. The alternative acquisition strategies with truncated k-space performed no better than the common AW approach. Compartment-based spectroscopy approaches provide an attractive reconstruction method for cardiac 31 P spectroscopy at 7 T, improving reproducibility and SNR without the need for a dedicated k-space sampling strategy.

Developing a metabolic clearance rate framework as a translational analysis approach for hyperpolarized 13C magnetic resonance imaging.

Hyperpolarized carbon-13 magnetic resonance imaging is a promising technique for in vivo metabolic interrogation of alterations between health and disease. This study introduces a formalism for quantifying the metabolic information in hyperpolarized imaging. This study investigated a novel perfusion formalism and metabolic clearance rate (MCR) model in pre-clinical stroke and in the healthy human brain. Simulations showed that the proposed model was robust to perturbations in T1, transmit B1, and kPL. A significant difference in ipsilateral vs contralateral pyruvate derived cerebral blood flow (CBF) was detected in rats (140 ± 2 vs 89 ± 6 mL/100 g/min, p < 0.01, respectively) and pigs (139 ± 12 vs 95 ± 5 mL/100 g/min, p = 0.04, respectively), along with an increase in fractional metabolism (26 ± 5 vs 4 ± 2%, p < 0.01, respectively) in the rodent brain. In addition, a significant increase in ipsilateral vs contralateral MCR (0.034 ± 0.007 vs 0.017 ± 0.02/s, p = 0.03, respectively) and a decrease in mean transit time (31 ± 8 vs 60 ± 2 s, p = 0.04, respectively) was observed in the porcine brain. In conclusion, MCR mapping is a simple and robust approach to the post-processing of hyperpolarized magnetic resonance imaging.

Assessing the effect of anesthetic gas mixtures on hyperpolarized 13 C pyruvate metabolism in the rat brain.

PURPOSE: To determine the effect of altering anesthetic oxygen protocols on measurements of cerebral perfusion and metabolism in the rodent brain. METHODS: Seven rats were anesthetized and underwent serial MRI scans with hyperpolarized [1-13 C]pyruvate and perfusion weighted imaging. The anesthetic carrier gas protocol used varied from 100:0% to 90:10% to 60:40% O2 :N2 O. Spectra were quantified with AMARES and perfusion imaging was processed using model-free deconvolution. A 1-way ANOVA was used to compare results across groups, with pairwise t tests performed with correction for multiple comparisons. Spearman's correlation analysis was performed between O2 % and MR measurements. RESULTS: There was a significant increase in bicarbonate:total 13 C carbon and bicarbonate:13 C pyruvate when moving between 100:0 to 90:10 and 100:0 to 60:40 O2 :N2 O % (0.02 ± 0.01 vs. 0.019 ± 0.005 and 0.02 ± 0.01 vs. 0.05 ± 0.02, respectively) and (0.04 ± 0.01 vs. 0.03 ± 0.01 and 0.04 ± 0.01 vs. 0.08 ± 0.02, respectively). There was a significant difference in 13 C pyruvate time to peak when moving between 100:0 to 90:10 and 100:0 to 60:40 O2 :N2 O % (13 ± 2 vs. 10 ± 1 and 13 ± 2 vs. 7.5 ± 0.5 s, respectively) as well as significant differences in cerebral blood flow (CBF) between gas protocols. Significant correlations between bicarbonate:13 C pyruvate and gas protocol (ρ = -0.47), mean transit time and gas protocol (ρ = 0.41) and 13 C pyruvate time-to-peak and cerebral blood flow (ρ = -0.54) were also observed. CONCLUSIONS: These results demonstrate that the detection and quantification of cerebral metabolism and perfusion is dependent on the oxygen protocol used in the anesthetized rodent brain.

Acute intermittent hypoxia drives hepatic de novo lipogenesis in humans and rodents.

Background and aims: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver condition. It is tightly associated with an adverse metabolic phenotype (including obesity and type 2 diabetes) as well as with obstructive sleep apnoea (OSA) of which intermittent hypoxia is a critical component. Hepatic de novo lipogenesis (DNL) is a significant contributor to hepatic lipid content and the pathogenesis of NAFLD and has been proposed as a key pathway to target in the development of pharmacotherapies to treat NAFLD. Our aim is to use experimental models to investigate the impact of hypoxia on hepatic lipid metabolism independent of obesity and metabolic disease. Methods: Human and rodent studies incorporating stable isotopes and hyperinsulinaemic euglycaemic clamp studies were performed to assess the regulation of DNL and broader metabolic phenotype by intermittent hypoxia. Cell-based studies, including pharmacological and genetic manipulation of hypoxia-inducible factors (HIF), were used to examine the underlying mechanisms. Results: Hepatic DNL increased in response to acute intermittent hypoxia in humans, without alteration in glucose production or disposal. These observations were endorsed in a prolonged model of intermittent hypoxia in rodents using stable isotopic assessment of lipid metabolism. Changes in DNL were paralleled by increases in hepatic gene expression of acetyl CoA carboxylase 1 and fatty acid synthase. In human hepatoma cell lines, hypoxia increased both DNL and fatty acid uptake through HIF-1α and -2α dependent mechanisms. Conclusions: These studies provide robust evidence linking intermittent hypoxia and the regulation of DNL in both acute and sustained in vivo models of intermittent hypoxia, providing an important mechanistic link between hypoxia and NAFLD.

Energetic Basis for Exercise-Induced Pulmonary Congestion in Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Transient pulmonary congestion during exercise is emerging as an important determinant of reduced exercise capacity in heart failure with preserved ejection fraction (HFpEF). We sought to determine whether an abnormal cardiac energetic state underpins this process. METHODS: We recruited patients across the spectrum of diastolic dysfunction and HFpEF (controls, n=11; type 2 diabetes, n=9; HFpEF, n=14; and severe diastolic dysfunction attributable to cardiac amyloidosis, n=9). Cardiac energetics were measured using phosphorus spectroscopy to define the myocardial phosphocreatine to ATP ratio. Cardiac function was assessed by cardiovascular magnetic resonance cine imaging and echocardiography and lung water using magnetic resonance proton density mapping. Studies were performed at rest and during submaximal exercise using a magnetic resonance imaging ergometer. RESULTS: Paralleling the stepwise decline in diastolic function across the groups (E/e' ratio; P<0.001) was an increase in NT-proBNP (N-terminal pro-brain natriuretic peptide; P<0.001) and a reduction in phosphocreatine/ATP ratio (control, 2.15 [2.09, 2.29]; type 2 diabetes, 1.71 [1.61, 1.91]; HFpEF, 1.66 [1.44, 1.89]; cardiac amyloidosis, 1.30 [1.16, 1.53]; P<0.001). During 20-W exercise, lower left ventricular diastolic filling rates (r=0.58; P<0.001), lower left ventricular diastolic reserve (r=0.55; P<0.001), left atrial dilatation (r=-0.52; P<0.001), lower right ventricular contractile reserve (right ventricular ejection fraction change, r=0.57; P<0.001), and right atrial dilation (r=-0.71; P<0.001) were all linked to lower phosphocreatine/ATP ratio. Along with these changes, pulmonary proton density mapping revealed transient pulmonary congestion in patients with HFpEF (+4.4% [0.5, 6.4]; P=0.002) and cardiac amyloidosis (+6.4% [3.3, 10.0]; P=0.004), which was not seen in healthy controls (-0.1% [-1.9, 2.1]; P=0.89) or type 2 diabetes without HFpEF (+0.8% [-1.7, 1.9]; P=0.82). The development of exercise-induced pulmonary congestion was associated with lower phosphocreatine/ATP ratio (r=-0.43; P=0.004). CONCLUSIONS: A gradient of myocardial energetic deficit exists across the spectrum of HFpEF. Even at low workload, this energetic deficit is related to markedly abnormal exercise responses in all 4 cardiac chambers, which is associated with detectable pulmonary congestion. The findings support an energetic basis for transient pulmonary congestion in HFpEF.

Maternal iron deficiency perturbs embryonic cardiovascular development in mice.

Congenital heart disease (CHD) is the most common class of human birth defects, with a prevalence of 0.9% of births. However, two-thirds of cases have an unknown cause, and many of these are thought to be caused by in utero exposure to environmental teratogens. Here we identify a potential teratogen causing CHD in mice: maternal iron deficiency (ID). We show that maternal ID in mice causes severe cardiovascular defects in the offspring. These defects likely arise from increased retinoic acid signalling in ID embryos. The defects can be prevented by iron administration in early pregnancy. It has also been proposed that teratogen exposure may potentiate the effects of genetic predisposition to CHD through gene-environment interaction. Here we show that maternal ID increases the severity of heart and craniofacial defects in a mouse model of Down syndrome. It will be important to understand if the effects of maternal ID seen here in mice may have clinical implications for women.

Rapid, B1 -insensitive, dual-band quasi-adiabatic saturation transfer with optimal control for complete quantification of myocardial ATP flux.

PURPOSE: Phosphorus saturation-transfer experiments can quantify metabolic fluxes noninvasively. Typically, the forward flux through the creatine kinase reaction is investigated by observing the decrease in phosphocreatine (PCr) after saturation of γ-ATP. The quantification of total ATP utilization is currently underexplored, as it requires simultaneous saturation of inorganic phosphate ( Pi ) and PCr. This is challenging, as currently available saturation pulses reduce the already-low γ-ATP signal present. METHODS: Using a hybrid optimal-control and Shinnar-Le Roux method, a quasi-adiabatic RF pulse was designed for the dual saturation of PCr and Pi to enable determination of total ATP utilization. The pulses were evaluated in Bloch equation simulations, compared with a conventional hard-cosine DANTE saturation sequence, before being applied to perfused rat hearts at 11.7 T. RESULTS: The quasi-adiabatic pulse was insensitive to a >2.5-fold variation in B1 , producing equivalent saturation with a 53% reduction in delivered pulse power and a 33-fold reduction in spillover at the minimum effective B1 . This enabled the complete quantification of the synthesis and degradation fluxes for ATP in 30-45 minutes in the perfused rat heart. While the net synthesis flux (4.24 ± 0.8 mM/s, SEM) was not significantly different from degradation flux (6.88 ± 2 mM/s, P = .06) and both measures are consistent with prior work, nonlinear error analysis highlights uncertainties in the Pi -to-ATP measurement that may explain a trend suggesting a possible imbalance. CONCLUSIONS: This work demonstrates a novel quasi-adiabatic dual-saturation RF pulse with significantly improved performance that can be used to measure ATP turnover in the heart in vivo.

Metabolic Effects of Doxorubicin on the Rat Liver Assessed With Hyperpolarized MRI and Metabolomics.

Doxorubicin (DOX) is a successful chemotherapeutic widely used for the treatment of a range of cancers. However, DOX can have serious side-effects, with cardiotoxicity and hepatotoxicity being the most common events. Oxidative stress and changes in metabolism and bioenergetics are thought to be at the core of these toxicities. We have previously shown in a clinically-relevant rat model that a low DOX dose of 2 mg kg-1 week-1 for 6 weeks does not lead to cardiac functional decline or changes in cardiac carbohydrate metabolism, assessed with hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopy (MRS). We now set out to assess whether there are any signs of liver damage or altered liver metabolism using this subclinical model. We found no increase in plasma alanine aminotransferase (ALT) activity, a measure of liver damage, following DOX treatment in rats at any time point. We also saw no changes in liver carbohydrate metabolism, using hyperpolarized [1-13C]pyruvate MRS. However, using metabolomic analysis of liver metabolite extracts at the final time point, we found an increase in most acyl-carnitine species as well as increases in high energy phosphates, citrate and markers of oxidative stress. This may indicate early signs of steatohepatitis, with increased and decompensated fatty acid uptake and oxidation, leading to oxidative stress.

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.

Nicotinic acid receptor agonists impair myocardial contractility by energy starvation.

Nicotinic acid receptor agonists have previously been shown to cause acute reductions in cardiac contractility. We sought to uncover the changes in cardiac metabolism underlying these alterations in function. In nine humans, we recorded cardiac energetics and function before and after a single oral dose of nicotinic acid using cardiac MRI to demonstrate contractile function and Phosphorus-31 (31 P) magnetic resonance spectroscopy to demonstrate myocardial energetics. Nicotinic Acid 400 mg lowered ejection fraction by 4% (64 ± 8% to 60 ± 7%, P = .03), and was accompanied by a fall in phosphocreatine/ATP ratio by 0.4 (2.2 ± 0.4 to 1.8 ± 0.1, P = .04). In four groups of eight Wistar rats, we used pyruvate dehydrogenase (PDH) flux studies to demonstrate changes in carbohydrate metabolism induced by the nicotinic acid receptor agonist, Acipimox, using hyperpolarized Carbon-13 (13 C) magnetic resonance spectroscopy. In rats which had been starved overnight, Acipimox caused a fall in ejection fraction by 7.8% (67.5 ± 8.9 to 60 ± 3.1, P = .03) and a nearly threefold rise in flux through PDH (from 0.182 ± 0.114 to 0.486 ± 0.139, P = .002), though this rise did not match pyruvate dehydrogenase flux observed in rats fed carbohydrate rich chow (0.726 ± 0.201). In fed rats, Acipimox decreased pyruvate dehydrogenase flux (to 0.512 ± 0.13, P = .04). Concentration of plasma insulin fell by two-thirds in fed rats administered Acipimox (from 1695 ± 891 ng/L to 550 ± 222 ng/L, P = .005) in spite of glucose concentrations remaining the same. In conclusion, we demonstrate that nicotinic acid receptor agonists impair cardiac contractility associated with a decline in cardiac energetics and show that the mechanism is likely a combination of reduced fatty acid availability and a failure to upregulate carbohydrate metabolism, essentially starving the heart of fuel.

Rescue of myocardial energetic dysfunction in diabetes through the correction of mitochondrial hyperacetylation by honokiol.

Cardiac energetic dysfunction has been reported in patients with type 2 diabetes (T2D) and is an independent predictor of mortality. Identification of the mechanisms driving mitochondrial dysfunction, and therapeutic strategies to rescue these modifications, will improve myocardial energetics in T2D. We demonstrate using 31P-magnetic resonance spectroscopy (31P-MRS) that decreased cardiac ATP and phosphocreatine (PCr) concentrations occurred before contractile dysfunction or a reduction in PCr/ATP ratio in T2D. Real-time mitochondrial ATP synthesis rates and state 3 respiration rates were similarly depressed in T2D, implicating dysfunctional mitochondrial energy production. Driving this energetic dysfunction in T2D was an increase in mitochondrial protein acetylation, and increased ex vivo acetylation was shown to proportionally decrease mitochondrial respiration rates. Treating T2D rats in vivo with the mitochondrial deacetylase SIRT3 activator honokiol reversed the hyperacetylation of mitochondrial proteins and restored mitochondrial respiration rates to control levels. Using 13C-hyperpolarized MRS, respiration with different substrates, and enzyme assays, we localized this improvement to increased glutamate dehydrogenase activity. Finally, honokiol treatment increased ATP and PCr concentrations and increased total ATP synthesis flux in the T2D heart. In conclusion, hyperacetylation drives energetic dysfunction in T2D, and reversing acetylation with the SIRT3 activator honokiol rescued myocardial and mitochondrial energetics in T2D.

Use of cardiac magnetic resonance to detect changes in metabolism in heart failure.

The heart has a massive adenosine triphosphate (ATP) requirement, produced from the oxidation of metabolic substrates such as fat and glucose. Magnetic resonance spectroscopy offers a unique opportunity to probe this biochemistry: 31Phosphorus spectroscopy can demonstrate the production of ATP and quantify levels of the transport molecule phosphocreatine while 13Carbon spectroscopy can demonstrate the metabolic fates of glucose in real time. These techniques allow the metabolic deficits in heart failure to be interrogated and can be a potential future clinical tool.

Cardiac Energetics in Patients With Aortic Stenosis and Preserved Versus Reduced Ejection Fraction.

BACKGROUND: Why some but not all patients with severe aortic stenosis (SevAS) develop otherwise unexplained reduced systolic function is unclear. We investigate the hypothesis that reduced creatine kinase (CK) capacity and flux is associated with this transition. METHODS: We recruited 102 participants to 5 groups: moderate aortic stenosis (ModAS) (n=13), SevAS, left ventricular (LV) ejection fraction ≥55% (SevAS-preserved ejection fraction, n=37), SevAS, LV ejection fraction <55% (SevAS-reduced ejection fraction, n=15), healthy volunteers with nonhypertrophied hearts with normal systolic function (normal healthy volunteer, n=30), and patients with nonhypertrophied, non-pressure-loaded hearts with normal systolic function undergoing cardiac surgery and donating LV biopsy (non-pressure-loaded heart biopsy, n=7). All underwent cardiac magnetic resonance imaging and 31P magnetic resonance spectroscopy for myocardial energetics. LV biopsies (AS and non-pressure-loaded heart biopsy) were analyzed for CK total activity, CK isoforms, citrate synthase activity, and total creatine. Mitochondria-sarcomere diffusion distances were calculated by using serial block-face scanning electron microscopy. RESULTS: In the absence of failure, CK flux was lower in the presence of AS (by 32%, P=0.04), driven primarily by reduction in phosphocreatine/ATP (by 17%, P<0.001), with CK kf unchanged (P=0.46). Although lowest in the SevAS-reduced ejection fraction group, CK flux was not different from the SevAS-preserved ejection fraction group (P>0.99). Accompanying the fall in CK flux, total CK and citrate synthase activities and the absolute activities of mitochondrial-type CK and CK-MM isoforms were also lower (P<0.02, all analyses). Median mitochondria-sarcomere diffusion distances correlated well with CK total activity (r=0.86, P=0.003). CONCLUSIONS: Total CK capacity is reduced in SevAS, with median values lowest in those with systolic failure, consistent with reduced energy supply reserve. Despite this, in vivo magnetic resonance spectroscopy measures of resting CK flux suggest that ATP delivery is reduced earlier, at the moderate AS stage, where LV function remains preserved. These findings show that significant energetic impairment is already established in moderate AS and suggest that a fall in CK flux is not by itself a necessary cause of transition to systolic failure. However, because ATP demands increase with AS severity, this could increase susceptibility to systolic failure. As such, targeting CK capacity and flux may be a therapeutic strategy to prevent and treat systolic failure in AS.

Hyperpolarized 13C MRI: A novel approach for probing cerebral metabolism in health and neurological disease.

Cerebral metabolism is tightly regulated and fundamental for healthy neurological function. There is increasing evidence that alterations in this metabolism may be a precursor and early biomarker of later stage disease processes. Proton magnetic resonance spectroscopy (1H-MRS) is a powerful tool to non-invasively assess tissue metabolites and has many applications for studying the normal and diseased brain. However, the technique has limitations including low spatial and temporal resolution, difficulties in discriminating overlapping peaks, and challenges in assessing metabolic flux rather than steady-state concentrations. Hyperpolarized carbon-13 magnetic resonance imaging is an emerging clinical technique that may overcome some of these spatial and temporal limitations, providing novel insights into neurometabolism in both health and in pathological processes such as glioma, stroke and multiple sclerosis. This review will explore the growing body of pre-clinical data that demonstrates a potential role for the technique in assessing metabolism in the central nervous system. There are now a number of clinical studies being undertaken in this area and this review will present the emerging clinical data as well as the potential future applications of hyperpolarized 13C magnetic resonance imaging in the brain, in both clinical and pre-clinical studies.

13C Pyruvate Transport Across the Blood-Brain Barrier in Preclinical Hyperpolarised MRI.

Hyperpolarised MRI with Dynamic Nuclear Polarisation overcomes the fundamental thermodynamic limitations of conventional magnetic resonance, and is translating to human studies with several early-phase clinical trials in progress including early reports that demonstrate the utility of the technique to observe lactate production in human brain cancer patients. Owing to the fundamental coupling of metabolism and tissue function, metabolic neuroimaging with hyperpolarised [1-13C]pyruvate has the potential to be revolutionary in numerous neurological disorders (e.g. brain tumour, ischemic stroke, and multiple sclerosis). Through the use of [1-13C]pyruvate and ethyl-[1-13C]pyruvate in naïve brain, a rodent model of metastasis to the brain, or porcine brain subjected to mannitol osmotic shock, we show that pyruvate transport across the blood-brain barrier of anaesthetised animals is rate-limiting. We show through use of a well-characterised rat model of brain metastasis that the appearance of hyperpolarized [1-13C]lactate production corresponds to the point of blood-brain barrier breakdown in the disease. With the more lipophilic ethyl-[1-13C]pyruvate, we observe pyruvate production endogenously throughout the entire brain and lactate production only in the region of disease. In the in vivo porcine brain we show that mannitol shock permeabilises the blood-brain barrier sufficiently for a dramatic 90-fold increase in pyruvate transport and conversion to lactate in the brain, which is otherwise not resolvable. This suggests that earlier reports of whole-brain metabolism in anaesthetised animals may be confounded by partial volume effects and not informative enough for translational studies. Issues relating to pyruvate transport and partial volume effects must therefore be considered in pre-clinical studies investigating neuro-metabolism in anaesthetised animals, and we additionally note that these same techniques may provide a distinct biomarker of blood-brain barrier permeability in future studies.

Noninvasive Immunometabolic Cardiac Inflammation Imaging Using Hyperpolarized Magnetic Resonance.

RATIONALE: Current cardiovascular clinical imaging techniques offer only limited assessment of innate immune cell-driven inflammation, which is a potential therapeutic target in myocardial infarction (MI) and other diseases. Hyperpolarized magnetic resonance (MR) is an emerging imaging technology that generates contrast agents with 10- to 20 000-fold improvements in MR signal, enabling cardiac metabolite mapping. OBJECTIVE: To determine whether hyperpolarized MR using [1-13C]pyruvate can assess the local cardiac inflammatory response after MI. METHODS AND RESULTS: We performed hyperpolarized [1-13C]pyruvate MR studies in small and large animal models of MI and in macrophage-like cell lines and measured the resulting [1-13C]lactate signals. MI caused intense [1-13C]lactate signal in healing myocardial segments at both day 3 and 7 after rodent MI, which was normalized at both time points after monocyte/macrophage depletion. A near-identical [1-13C]lactate signature was also seen at day 7 after experimental MI in pigs. Hyperpolarized [1-13C]pyruvate MR spectroscopy in macrophage-like cell suspensions demonstrated that macrophage activation and polarization with lipopolysaccharide almost doubled hyperpolarized lactate label flux rates in vitro; blockade of glycolysis with 2-deoxyglucose in activated cells normalized lactate label flux rates and markedly inhibited the production of key proinflammatory cytokines. Systemic administration of 2-deoxyglucose after rodent MI normalized the hyperpolarized [1-13C]lactate signal in healing myocardial segments at day 3 and also caused dose-dependent improvement in IL (interleukin)-1ß expression in infarct tissue without impairing the production of key reparative cytokines. Cine MRI demonstrated improvements in systolic function in 2-DG (2-deoxyglucose)-treated rats at 3 months. CONCLUSIONS: Hyperpolarized MR using [1-13C]pyruvate provides a novel method for the assessment of innate immune cell-driven inflammation in the heart after MI, with broad potential applicability across other cardiovascular disease states and suitability for early clinical translation.

Cardiac applications of hyperpolarised magnetic resonance.

Cardiovascular disease is the leading cause of death world-wide. It is increasingly recognised that cardiac pathologies show, or may even be caused by, changes in metabolism, leading to impaired cardiac energetics. The heart turns over 15 times its own weight in ATP every day and thus relies heavily on the availability of substrates and on efficient oxidation to generate this ATP. A number of old and emerging drugs that target different aspects of metabolism are showing promising results with regard to improved cardiac outcomes in patients. A non-invasive imaging technique that could assess the role of different aspects of metabolism in heart disease, as well as measure changes in cardiac energetics due to treatment, would be valuable in the routine clinical care of cardiac patients. Hyperpolarised magnetic resonance spectroscopy and imaging have revolutionised metabolic imaging, allowing real-time metabolic flux assessment in vivo for the first time. In this review we summarise metabolism in the healthy and diseased heart, give an introduction to the hyperpolarisation technique, 'dynamic nuclear polarisation' (DNP), and review the preclinical studies that have thus far explored healthy cardiac metabolism and different models of human heart disease. We furthermore show what advances have been made to translate this technique into the clinic, what technical challenges still remain and what unmet clinical needs and unexplored metabolic substrates still need to be assessed by researchers in this exciting and fast-moving field.

Hyperpolarized [1,4-13C2]Fumarate Enables Magnetic Resonance-Based Imaging of Myocardial Necrosis.

OBJECTIVES: The aim of this study was to determine if hyperpolarized [1,4-13C2]malate imaging could measure cardiomyocyte necrosis after myocardial infarction (MI). BACKGROUND: MI is defined by an acute burst of cellular necrosis and the subsequent cascade of structural and functional adaptations. Quantifying necrosis in the clinic after MI remains challenging. Magnetic resonance-based detection of the conversion of hyperpolarized [1,4-13C2]fumarate to [1,4-13C2]malate, enabled by disrupted cell membrane integrity, has measured cellular necrosis in vivo in other tissue types. Our aim was to determine whether hyperpolarized [1,4-13C2]malate imaging could measure necrosis after MI. METHODS: Isolated perfused hearts were given hyperpolarized [1,4-13C2]fumarate at baseline, immediately after 20 min of ischemia, and after 45 min of reperfusion. Magnetic resonance spectroscopy measured conversion into [1,4-13C2]malate. Left ventricular function and energetics were monitored throughout the protocol, buffer samples were collected and hearts were preserved for further analyses. For in vivo studies, magnetic resonance spectroscopy and a novel spatial-spectral magnetic resonance imaging sequence were implemented to assess cardiomyocyte necrosis in rats, 1 day and 1 week after cryo-induced MI. RESULTS: In isolated hearts, [1,4-13C2]malate production became apparent after 45 min of reperfusion, and increased 2.7-fold compared with baseline. Expression of dicarboxylic acid transporter genes were negligible in healthy and reperfused hearts, and lactate dehydrogenase release and infarct size were significantly increased in reperfused hearts. Nonlinear regression revealed that [1,4-13C2]malate production was induced when adenosine triphosphate was depleted by >50%, below 5.3 mmol/l (R2 = 0.904). In vivo, the quantity of [1,4-13C2]malate visible increased 82-fold over controls 1 day after infarction, maintaining a 31-fold increase 7 days post-infarct. [1,4-13C2]Malate could be resolved using hyperpolarized magnetic resonance imaging in the infarct region one day after MI; [1,4-13C2]malate was not visible in control hearts. CONCLUSIONS: Malate production in the infarcted heart appears to provide a specific probe of necrosis acutely after MI, and for at least 1 week afterward. This technique could offer an alternative noninvasive method to measure cellular necrosis in heart disease, and warrants further investigation in patients.

Simultaneous assessment of cardiac metabolism and perfusion using copolarized [1-13 C]pyruvate and 13 C-urea.

PURPOSE: To demonstrate the feasibility of imaging a bolus of co-polarized [1-13 C]pyruvate and 13 C-urea to simultaneously assess both metabolism and perfusion in the rodent heart. METHODS: Copolarized [1-13 C]pyruvate and 13 C-urea was imaged using a multi-echo, flow-sensitized spiral pulse sequence. Healthy rats were scanned in a two-factor factorial design (n = 12 total; metabolism: overnight fasting versus fed with dichloroacetate injection; perfusion: rest versus adenosine stress-induced hyperemia). RESULTS: Alterations in metabolism were detected by changes in pyruvate metabolism into 13 C-bicarbonate. Statistically independent alterations in perfusion were detected by changes in myocardial pyruvate and urea signals. CONCLUSION: The new pulse sequence was used to obtain maps of metabolism and perfusion in the rodent heart in a single acquisition. This hyperpolarized 13 C imaging test is expected to enable new studies in which the cardiac metabolism/perfusion mismatch can be studied in the acute environment. Magn Reson Med 77:151-158, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine.