Dotarem

CMR was performed using either a Magnetom Aera 1.5T or Prisma 3.0T system (Siemens Healthineers, Erlangen, Germany). A standard CMR protocol was used including cine images, stress and rest perfusion and late gadolinium enhancement.21 (link) All subjects abstained from caffeine for at least 12 hours. Adenosine was infused for 4 min at 140 µg/kg/min (increased to 175 µg/kg/min if there was no heart rate response and symptoms). At peak vasodilator stress a gadolinium-based contrast agent (Dotarem, Guerbet, Paris, France) was injected at a dose of 0.05 mmol/kg at a rate of 4 mL/s. Three short axis slices (base, mid and apex) were acquired during the first pass of contrast (60 measurements). The acquisition was repeated at rest, with the short axis cine stack acquired between stress and rest.
Perfusion mapping was performed automatically and inline as previously described.15 (link) In brief, this was a single-bolus, dual-sequence technique with a balanced steady-state free precession (bSSFP) pulse sequence readout. LGE images were acquired in long axis and short axis using a free-breathing bright blood single-shot bSSFP sequence with phase-sensitive inversion recovery reconstruction and motion correction. Sequence details are provided in the supplementary appendix.

A HyperSense™ DNP polarizer (Oxford Instruments, Abingdon, UK) was used to polarize the ¹³C probes as described previously [21 (link)]. Twenty-four micro liters of neat [1-¹³C] pyruvic acid (Isotec Stable Isotopes, Miamisburg, OH, USA) with 16.5 mM trityl radical [tris(8-carboxy-2,2,6,6,-tetra(methoxyethyl)benzo[1,2-d:4,5-d’]bis(1,3)dithiole-4-yl)methyl sodium salt] (GE healthcare, Waukesha, WI, USA) and 1.5 mM Dotarem^® (Guerbet) and 55 µL [¹³C] urea (6.4 M in glycerol, (Isotec Stable Isotopes, Miamisburg, OH, USA) with 17.5 mM trityl radical OX63 (Oxford Instruments) and 0.2 mM Dotarem^® were co-polarized. Optimum polarization was achieved by adding the urea and pyruvic acid solutions to a sample cup separately and freezing them rapidly in a liquid nitrogen bath to form two separate glass layers, as previously described [21 (link)]. This was followed by dissolution in 4.5 mL of buffer containing 40 mM Tris, 80 mM NaOH, and 0.3 mM Na₂EDTA. The resulting dissolution mixture contained 80 mM [1-¹³C] pyruvic acid and 74 mM [¹³C] urea with average polarizations of 24 ± 5% and 18 ± 6%, respectively, and an average pH of 7.0 ± 0.5.

CMR was performed on a 3.0-T scanner (Ingenia, Philips Medical Systems). The CMR protocol included a cine short-axis stack for LV volumes, mass, and function assessment using a standard steady-state free-precession (SSFP) sequence. T2 mapping was performed using a free-breathing navigator-gated black-blood prepared gradient and spin-echo (GraSE) hybrid sequence in three short-axis slices (basal, midventricular, and apical) [11 (link)]. T1 mapping was performed using a 5 s(3 s)3 s MOLLI sequence with typical imaging parameters: voxel size 2 × 2 × 10 mm³, echo time = 0.7 ms, time to repetition = 2.3 ms, partial echo factor = 0.8, flip angle = 35°, SENSE factor = 2, linear phase encoding, ten start-up cycles to approach steady-state prior to imaging, typical effective inversion times between 134 and 5500 ms [16 (link)]. Ten minutes after injection of 0.15 mmol/kg gadoterate meglumine (Dotarem®, Guerbet), end-diastolic late gadolinium enhancement (LGE) images were acquired using phase-sensitive inversion recovery (PSIR) sequences in short-axis and two-, three-, and four-chamber views [12 (link)]. Details are in Supplemental Material.

A small group of mice were anesthetized at PND19 using 4% isofluorane in air and sacrificed by decapitation. The entire heads were placed in tubes containing a cold solution of 4% Paraformaldehyde (PFA) and 8 mM Gadolium (Dotarem 0.5 mmol/ml, Guerbet). The tubes were then left at 4°C for 6–12 weeks for the contrast agent to fully diffuse and stabilize in tissue prior to ex-vivo imaging.
Magnetic Resonance Imaging (MRI) was performed on a 3T preclinical horizontal bore scanner (MR Solutions, Guilford, UK), equipped with a quadrature volume coil designed for rodent head imaging, with a 17 cm horizontal bore. On analysis day, the heads were placed in a custom-made 3D holder filled with fluorinert (3M, MN, USA) and anatomical and structural MRI sequences were acquired. Three dimensional Fast Spin Echo (FSE3D) anatomical series were used to calculate the volumes of brain sub-regions. Spin Echo Diffusion Tensor Imaging (SE-DTI) was used to calculate the diffusions parameters as described below. The details of each sequence have been reported in the Supplementary Data 6. The 3D Brain Atlas (Invicro's VivoQuant™) provided 14 sub-regions and the analysis of the changes in brain volume or in the diffusion parameters was performed with Matlab 2020b (MathWorks, MA, USA). The atlas provided 14 sub-regions and the analysis was completed for all sub-regions.