CysPresso: a classification model utilizing deep learning protein representations to predict recombinant expression of cysteine-dense peptides.

BACKGROUND: Cysteine-dense peptides (CDPs) are an attractive pharmaceutical scaffold that display extreme biochemical properties, low immunogenicity, and the ability to bind targets with high affinity and selectivity. While many CDPs have potential and confirmed therapeutic uses, synthesis of CDPs is a challenge. Recent advances have made the recombinant expression of CDPs a viable alternative to chemical synthesis. Moreover, identifying CDPs that can be expressed in mammalian cells is crucial in predicting their compatibility with gene therapy and mRNA therapy. Currently, we lack the ability to identify CDPs that will express recombinantly in mammalian cells without labour intensive experimentation. To address this, we developed CysPresso, a novel machine learning model that predicts recombinant expression of CDPs based on primary sequence. RESULTS: We tested various protein representations generated by deep learning algorithms (SeqVec, proteInfer, AlphaFold2) for their suitability in predicting CDP expression and found that AlphaFold2 representations possessed the best predictive features. We then optimized the model by concatenation of AlphaFold2 representations, time series transformation with random convolutional kernels, and dataset partitioning. CONCLUSION: Our novel model, CysPresso, is the first to successfully predict recombinant CDP expression in mammalian cells and is particularly well suited for predicting recombinant expression of knottin peptides. When preprocessing the deep learning protein representation for supervised machine learning, we found that random convolutional kernel transformation preserves more pertinent information relevant for predicting expressibility than embedding averaging. Our study showcases the applicability of deep learning-based protein representations, such as those provided by AlphaFold2, in tasks beyond structure prediction.

Relationship between apparent diffusion coefficient and survival as a function of distance from gross tumor volume on radiation planning MRI in newly diagnosed glioblastoma.

PURPOSE: To investigate the changes in apparent diffusion coefficient (ADC) within incrementally-increased margins beyond the gross tumor volume (GTV) on post-operative radiation planning MRI and their prognostic utility in glioblastoma. METHODS: Radiation planning MRIs of adult patients with newly diagnosed glioblastoma from 2017 to 2020 were assessed. The ADC values were normalized to contralateral normal white matter (nADC). Using 1 mm isotropic incremental margin increases from the GTV, the nADC values were calculated at each increment. Age, ECOG performance status, extent of resection and MGMT promoter methylation status were obtained from medical records. Using univariate and multivariable Cox regression analysis, association of nADC to progression-free and overall survival (PFS, OS) was assessed at each increment. RESULTS: Seventy consecutive patients with mean age of 53.6 ± 10.3 years, were evaluated. The MGMT promoter was methylated in 31 (44.3%), unmethylated in 36 (51.6%) and unknown in 3 (4.3%) patients. 11 (16%) underwent biopsy, 41 (44%) subtotal resection and 18 (26%) gross total resection. For each 1 mm increase in distance from GTV, the nADC decreased by 0.16% (p < 0.0001). At 1-5 mm increment, the nADC was associated with OS (p < 0.01). From 6 to 11 mm increment the nADC was associated with OS with the p-value gradually increasing from 0.018 to 0.046. nADC was not associated with PFS. CONCLUSION: The nADC values at 1-11 mm increments from the GTV margin were associated with OS. Future prospective multicenter studies are needed to validate the findings and to pave the way for the utilization of ADC for margin reduction in radiation planning.

Putaminal Recombinant Glucocerebrosidase Delivery with Magnetic Resonance-Guided Focused Ultrasound in Parkinson's Disease: A Phase I Study.

BACKGROUND: GBA1 mutation is the most common genetic risk factor for Parkinson's disease (PD). Replacement of the lysosomal enzyme glucocerebrosidase (GCase) slows neurodegeneration in PD models and may be a promising disease-modifying therapy in patients with PD. However, recombinant GCase has limited penetration through the blood-brain barrier (BBB). Microbubble-mediated magnetic resonance-guided focused ultrasound (MRgFUS) can reversibly disrupt the BBB for drug delivery. METHODS: This open-label phase I study investigated the safety and feasibility of MRgFUS putaminal delivery of intravenous GCase at escalating doses (15 to 30 to 60 IU/kg) every 2 weeks in four patients with PD with GBA1 mutations. RESULTS: BBB permeability was achieved and restored in all patients as quantified by dynamic contrast-enhanced magnetic resonance imaging after treatment. There were no serious adverse events. Two patients developed transient dyskinesia after treatment. Blinded Movement Disorder Society-Unified Parkinson's Disease Rating Scale motor scores off medication decreased by 12% at 6 months from baseline (from 26 ± 9 to 22 ± 6). Standardized uptake value ratio on fluorodeoxyglucose positron emission tomography imaging in the treated putamen reduced from 1.66 ± 0.14 to 1.27 ± 0.08. CONCLUSIONS: Results from this study demonstrate the safety and feasibility of MRgFUS GCase delivery in PD and support further investigation of this approach. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

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.

Predicting survival in patients with glioblastoma using MRI radiomic features extracted from radiation planning volumes.

BACKGROUND: Quantitative image analysis using pre-operative magnetic resonance imaging (MRI) has been able to predict survival in patients with glioblastoma (GBM). The study explored the role of postoperative radiation (RT) planning MRI-based radiomics to predict the outcomes, with features extracted from the gross tumor volume (GTV) and clinical target volume (CTV). METHODS: Patients with IDH-wildtype GBM treated with adjuvant RT having MRI as a part of RT planning process were included in the study. 546 features were extracted from each GTV and CTV. A LASSO Cox model was applied, and internal validation was performed using leave-one-out cross-validation with overall survival as endpoint. Cross-validated time-dependent area under curve (AUC) was constructed to test the efficacy of the radiomics model, and clinical features were used to generate a combined model. Analysis was done for the entire group and in individual surgical groups-gross total excision (GTR), subtotal resection (STR), and biopsy. RESULTS: 235 patients were included in the study with 57, 118, and 60 in the GTR, STR, and biopsy subgroup, respectively. Using the radiomics model, binary risk groups were feasible in the entire cohort (p < 0.01) and biopsy group (p = 0.04), but not in the other two surgical groups individually. The integrated AUC (iAUC) was 0.613 for radiomics-based classification in the biopsy subgroup, which improved to 0.632 with the inclusion of clinical features. CONCLUSION: Imaging features extracted from the GTV and CTV regions can lead to risk-stratification of GBM undergoing biopsy, while the utility in other individual subgroups needs to be further explored.

Cardiac metabolic imaging using hyperpolarized [1-13 C]lactate as a substrate.

Hyperpolarized (HP) [1-13 C]lactate is an attractive alternative to [1-13 C]pyruvate as a substrate to investigate cardiac metabolism in vivo: it can be administered safely at a higher dose and can be polarized to a degree similar to pyruvate via dynamic nuclear polarization. While 13 C cardiac experiments using HP lactate have been performed in small animal models, they have not been demonstrated in large animal models or humans. Utilizing the same hardware and data acquisition methods as the first human HP 13 C cardiac study, 13 C metabolic images were acquired following injections of HP [1-13 C]lactate in porcine hearts. Data were also acquired using HP [1-13 C]pyruvate for comparison. The 13 C bicarbonate signal was localized to the myocardium and had a similar appearance with both substrates for all animals. No 13 C pyruvate signal was detected in the experiments following injection of HP 13 C lactate. The signal-to-noise ratio (SNR) of injected lactate was 88 ± 4% of the SNR of injected pyruvate, and the SNR of bicarbonate in the experiments using lactate as the substrate was 52 ± 19% of the SNR in the experiments using pyruvate as the substrate. The lower SNR was likely due to the shorter T1 of [1-13 C]lactate as compared with [1-13 C]pyruvate and the additional enzyme-catalyzed metabolic conversion step before the 13 C nuclei from [1-13 C]lactate were detected as 13 C bicarbonate. While challenges remain, the potential of HP lactate as a substrate for clinical metabolic imaging of human heart has been demonstrated.

Deuterium MRS of early treatment-induced changes in tumour lactate in vitro.

Elevated production of lactate is a key characteristic of aberrant tumour cell metabolism and can be non-invasively measured as an early marker of tumour response using deuterium (2 H) MRS. Following treatment, changes in the 2 H-labelled lactate signal could identify tumour cell death or impaired metabolic function, which precede morphological changes conventionally used to assess tumour response. In this work, the association between apoptotic cell death, extracellular lactate concentration, and early treatment-induced changes in the 2 H-labelled lactate signal was established in an in vitro tumour model. Experiments were conducted at 7 T on acute myeloid leukaemia (AML) cells, which had been treated with 10 µg/mL of the chemotherapeutic agent cisplatin. At 24 and 48 h after cisplatin treatment the cells were supplied with 20 mM of [6,6'-2 H2 ]glucose and scanned over 2 h using a two-dimensional 2 H MR spectroscopic imaging sequence. The resulting signals from 2 H-labelled glucose, lactate, and water were quantified using a spectral fitting algorithm implemented on the Oxford Spectroscopy Analysis MATLAB toolbox. After scanning, the cells were processed for histological stains (terminal deoxynucleotidyl transferase UTP nick end labelling and haematoxylin and eosin) to assess apoptotic area fraction and cell morphology respectively, while a colorimetric assay was used to measure extracellular lactate concentrations in the supernatant. Significantly lower levels of 2 H-labelled lactate were observed in the 48 h treated cells compared with the untreated and 24 h treated cells, and these changes were significantly correlated with an increase in apoptotic fraction and a decrease in extracellular lactate. By establishing the biological processes associated with treatment-induced changes in the 2 H-labelled lactate signal, these findings suggest that 2 H MRS of lactate may be valuable in evaluating early tumour response.

MRI radiomics to differentiate between low grade glioma and glioblastoma peritumoral region.

BACKGROUND: The peritumoral region (PTR) of glioblastoma (GBM) appears as a T2W-hyperintensity and is composed of microscopic tumor and edema. Infiltrative low grade glioma (LGG) comprises tumor cells that seem similar to GBM PTR on MRI. The work here explored if a radiomics-based approach can distinguish between the two groups (tumor and edema versus tumor alone). METHODS: Patients with GBM and LGG imaged using a 1.5 T MRI were included in the study. Image data from cases of GBM PTR, and LGG were manually segmented guided by T2W hyperintensity. A set of 91 first-order and texture features were determined from each of T1W-contrast, and T2W-FLAIR, diffusion-weighted imaging sequences. Applying filtration techniques, a total of 3822 features were obtained. Different feature reduction techniques were employed, and a subsequent model was constructed using four machine learning classifiers. Leave-one-out cross-validation was used to assess classifier performance. RESULTS: The analysis included 42 GBM and 36 LGG. The best performance was obtained using AdaBoost classifier using all the features with a sensitivity, specificity, accuracy, and area of curve (AUC) of 91%, 86%, 89%, and 0.96, respectively. Amongst the feature selection techniques, the recursive feature elimination technique had the best results, with an AUC ranging from 0.87 to 0.92. Evaluation with the F-test resulted in the most consistent feature selection with 3 T1W-contrast texture features chosen in over 90% of instances. CONCLUSIONS: Quantitative analysis of conventional MRI sequences can effectively demarcate GBM PTR from LGG, which is otherwise indistinguishable on visual estimation.

Quantitative mapping of individual voxels in the peritumoral region of IDH-wildtype glioblastoma to distinguish between tumor infiltration and edema.

PURPOSE: The peritumoral region (PTR) in glioblastoma (GBM) represents a combination of infiltrative tumor and vasogenic edema, which are indistinguishable on magnetic resonance imaging (MRI). We developed a radiomic signature by using imaging data from low grade glioma (LGG) (marker of tumor) and PTR of brain metastasis (BM) (marker of edema) and applied it on the GBM PTR to generate probabilistic maps. METHODS: 270 features were extracted from T1-weighted, T2-weighted, and apparent diffusion coefficient maps in over 3.5 million voxels of LGG (36 segments) and BM (45 segments) scanned in a 1.5T MRI. A support vector machine classifier was used to develop the radiomics model from approximately 50% voxels (downsampled to 10%) and validated with the remaining. The model was applied to over 575,000 voxels of the PTR of 10 patients with GBM to generate a quantitative map using Platt scaling (infiltrative tumor vs. edema). RESULTS: The radiomics model had an accuracy of 0.92 and 0.79 in the training and test set, respectively (LGG vs. BM). When extrapolated on the GBM PTR, 9 of 10 patients had a higher percentage of voxels with a tumor-like signature over radiological recurrence areas. In 7 of 10 patients, the areas under curves (AUC) were > 0.50 confirming a positive correlation. Including all the voxels from the GBM patients, the infiltration signature had an AUC of 0.61 to predict recurrence. CONCLUSION: A radiomic signature can demarcate areas of microscopic tumors from edema in the PTR of GBM, which correlates with areas of future recurrence.

Quantitative CEST and MT at 1.5T for monitoring treatment response in glioblastoma: early and late tumor progression during chemoradiation.

PURPOSE: Quantitative MRI (qMRI) was performed using a 1.5T protocol that includes a novel chemical exchange saturation transfer/magnetization transfer (CEST/MT) approach. The purpose of this prospective study was to determine if qMRI metrics at baseline, at the 10th and 20th fraction during a 30 fraction/6 week standard chemoradiation (CRT) schedule, and at 1 month following treatment could be an early indicator of response for glioblastoma (GBM). METHODS: The study included 51 newly diagnosed GBM patients. Four regions-of-interest (ROI) were analyzed: (i) the radiation defined clinical target volume (CTV), (ii) radiation defined gross tumor volume (GTV), (iii) enhancing-tumor regions, and (iv) FLAIR-hyperintense regions. Quantitative CEST, MT, T1 and T2 parameters were compared between those patients progressing within 6.9 months (early), and those progressing after CRT (late), using mixed modelling. Exploratory predictive modelling was performed to identify significant predictors of early progression using a multivariable LASSO model. RESULTS: Results were dependent on the specific tumor ROI analyzed and the imaging time point. The baseline CEST asymmetry within the CTV was significantly higher in the early progression cohort. Other significant predictors included the T2 of the MT pools (for semi-solid at fraction 20 and water at 1 month after CRT), the exchange rate (at fraction 20) and the MGMT methylation status. CONCLUSIONS: We observe the potential for multiparametric qMRI, including a novel pulsed CEST/MT approach, to show potential in distinguishing early from late progression GBM cohorts. Ultimately, the goal is to personalize therapeutic decisions and treatment adaptation based on non-invasive imaging-based biomarkers.

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.

Quantification of pulsed saturation transfer at 1.5T and 3T.

PURPOSE: To compare magnetization transfer (MT) and CEST effects between 1.5T and 3T in phantom and in vivo experiments. METHODS: A pulsed saturation scheme using block-shaped pulses separated by gaps was used to overcome the single RF amplifier duty cycle limitations of a clinical 1.5T scanner. Modeling was performed by incorporating the extended phase graph formalism into a Bloch-McConnell simulation. Two saturation pulse types (with long and short pulses) were used. Estimated parameters for MT (the semi-solid pool fraction, M0B ; the semi-solid transverse relaxation time, T2B ) and CEST (asymmetry; areas) were compared between 1.5T and 3T in phantoms and in the healthy brain. RESULTS: Improved fits were shown after inclusion of extended phase graphs. Semi-solid pool fractions in phantom (for agar with ammonium chloride) were higher for short compared to long pulses at 3T (by 19% over all concentrations) and higher at 1.5T compared to 3T (by 5%) using short pulses. In the in vivo experiments, differentiation of white and gray matter was seen in the brain at both field strengths with improved white-gray matter contrast at 3T. In white matter, the mean semi-solid fractions were 18 ± 2% at 3T and 15 ± 2% at 1.5T. The CEST asymmetry in white matter was negative (-4.9 ± 0.4%) at 3T and zero (0.0 ± 0.3%) at 1.5T. CONCLUSIONS: The pulsed saturation method with short pulses, using the extended phase graph formalism in the Bloch McConnell simulations, led to improved model fits to the data, when compared to those without extended phase graphs.

Evaluating the accuracy of multicomponent T2 parameters for luminal water imaging of the prostate with acceleration using inner-volume 3D GRASE.

PURPOSE: Prostate cancer can be detected using a multicomponent T2 mapping technique termed luminal water imaging. The purpose of this study is twofold: 1) To accelerate the luminal water imaging acquisition by using inner volume selection as part of a gradient and spin echo sequence, and 2) to evaluate the accuracy of luminal water fractions and multicomponent T2 relaxation times. METHODS: The accuracy of parameter estimates was assessed using Monte Carlo simulations, in phantom experiments and in the prostate (in 5 healthy subjects). Two fitting methods, nonnegative least squares and biexponential fitting with stimulated echo correction, were compared. RESULTS: Results demonstrate that inner volume selection in a gradient and spin echo sequence is effective for accelerating prostate luminal water imaging by at least threefold. Evaluation of the accuracy shows that the estimated luminal water fractions are relatively accurate, but the short- and long-T2 relaxation times should be interpreted with caution in noisy scenarios (SNR < 100) and when the corresponding fractions are small ( < 0.5). The mean luminal water fractions obtained at SNR above 100 are 0.27 ± 0.07 for the peripheral zone for both fitting methods, 0.16 ± 0.04 for the transition zone with nonnegative least squares, and 0.16 ± 0.03 for the transition zone with biexponential fitting including stimulated echo correction. CONCLUSION: The shortened scan duration allows the luminal water imaging sequence to be easily integrated into a standard multiparametric prostate MRI protocol.

Susceptibility-induced distortion correction in hyperpolarized echo planar imaging.

PURPOSE: Echo planar imaging is an attractive rapid imaging readout that can image hyperpolarized compounds in vivo. By alternating the sign of the phase encoding gradient waveform, spatial offsets arising from uncertain frequency shifts can be determined. We show here that blip-reversed echo planar imaging can also be used to correct for susceptibility and B0 inhomogeneity effects that would otherwise produce image-domain distortion in the heart. METHODS: Previously acquired blip-reversed cardiac 3D-Spectral-Spatial echo planar imaging volumetric timecourses of hyperpolarized [1-13 C]pyruvate were distortion corrected by a deformation field estimated by reconstructing signal-to-noise ratio (SNR)-weighted progressively subsampled temporally summed images of each metabolite. RESULTS: Reconstructing blip-reversed data as proposed produced volumetric timecourses that overlaid with proton reference images more consistently than without such corrections. CONCLUSION: The method proposed may form an attractive method to correct for image-domain distortions in hyperpolarized echo planar imaging experiments. Magn Reson Med 79:2135-2141, 2018. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Simultaneous multislice acquisition without trajectory modification for hyperpolarized 13 C experiments.

PURPOSE: To investigate the feasibility of performing large FOV hyperpolarized 13 C metabolic imaging using simultaneous multislice excitation. METHODS: A spectral-spatial multislice excitation pulse was constructed by cosine modulation and incorporated into a 13 C spiral imaging sequence. Phantom and in vivo pig experiments were performed to test the feasibility of simultaneous multislice data acquisition and image reconstruction. In vivo cardiac-gated images of hyperpolarized pyruvate, bicarbonate, and lactate were obtained at 1 × 1 × 1 cm3 resolution over a 48 × 48 × 24 cm3 FOV with 2-fold acceleration in the slice direction. Sensitivity encoding was used for image reconstruction with both autocalibrated and numerically calculated coil sensitivities. RESULTS: Simultaneous multislice images obtained with 2-fold acceleration were comparable to reference unaccelerated images. Retained SNR figures greater than 80% were achieved over the part of the image containing the heart. CONCLUSION: This method is anticipated to enable large FOV imaging studies using hyperpolarized 13 C substrates, with an aim toward whole-body exams that have to date been out of reach.

Hyperpolarized ketone body metabolism in the rat heart.

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

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.

Mapping of intracellular pH in the in vivo rodent heart using hyperpolarized [1-13C]pyruvate.

PURPOSE: To demonstrate the feasibility of mapping intracellular pH within the in vivo rodent heart. Alterations in cardiac acid-base balance can lead to acute contractile depression and alterations in Ca2+ signaling. The transient reduction in adenosine triphosphate (ATP) consumption and cardiac contractility may be initially beneficial; however, sustained pH changes can be maladaptive, leading to myocardial damage and electrical arrhythmias. METHODS: Spectrally selective radiofrequency (RF) pulses were used to excite the HCO3- and CO2 resonances individually while preserving signal from the injected hyperpolarized [1-13 C]pyruvate. The large flip angle pulses were placed within a three-dimensional (3D) imaging acquisition, which exploited CA-mediated label exchange between HCO3- and CO2 . Images at 4.5 × 4.5 × 5 mm3 resolution were obtained in the in vivo rodent heart. The technique was evaluated in healthy rodents scanned at baseline and during high cardiac workload induced by dobutamine infusion. RESULTS: The intracellular pH was measured to be 7.15 ± 0.04 at baseline, and decreased to 6.90 ± 0.06 following 15 min of continuous ß-adrenergic stimulation. CONCLUSIONS: Volumetric maps of intracellular pH can be obtained following an injection of hyperpolarized [1-13 C]pyruvate. The new method is anticipated to enable assessment of stress-inducible ischemia and potential ventricular arrythmogenic substrates within the ischemic heart. Magn Reson Med 77:1810-1817, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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.

Cardiac perfusion imaging using hyperpolarized (13)C urea using flow sensitizing gradients.

PURPOSE: To demonstrate the feasibility of imaging the first passage of a bolus of hyperpolarized (13)C urea through the rodent heart using flow-sensitizing gradients to reduce signal from the blood pool. METHODS: A flow-sensitizing bipolar gradient was optimized to reduce the bright signal within the cardiac chambers, enabling improved contrast of the agent within the tissue capillary bed. The gradient was incorporated into a dynamic golden angle spiral (13)C imaging sequence. Healthy rats were scanned during rest (n = 3) and under adenosine stress-induced hyperemia (n = 3). RESULTS: A two-fold increase in myocardial perfusion relative to rest was detected during adenosine stress-induced hyperemia, consistent with a myocardial perfusion reserve of two in rodents. CONCLUSION: The new pulse sequence was used to obtain dynamic images of the first passage of hyperpolarized (13)C urea in the rodent heart, without contamination from bright signal within the neighboring cardiac lumen. This probe of myocardial perfusion is expected to enable new hyperpolarized (13)C studies in which the cardiac metabolism/perfusion mismatch can be identified. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance.