Diffusion tensor imaging (DTI) (1) has emerged as the method of choice for noninvasive quantifications of myocardial microstructure, which play an essential role in the structural and functional properties of the heart. However, the origins and behaviors of DTI measurements as functions of myocardial remodeling (e.g., myocytes growth) during development and in disease remain poorly known or understood. In this work, conventional and bi-compartmental DTI and direct histological correlation were performed on an animal model of myocardial development (2) to investigate the effects of tissue remodeling.Hearts were obtained from 120 d and 130 d post-gestation, full term (150 d) at birth, and term-born 5 month-old lambs (n=6-8 each), arrested at cardiac diastole and fixed by perfusion using KCl and formalin as described previously (3). DTI was conducted on a Bruker Biospec 7T scanner using multi-slice diffusion-weighted spin echo (SE) sequence (1500 ms TR, 30 ms TE, 64x64 matrix, 2.0 mm thickness, 4-7 cm² FOV depending on heart size, 12 encoding directions with 5 and 15 ms pulse width and separation, 1500 s/mm² b-value). Signal to noise ratio was above 80 for all B0 scans. Diffusion tensors were estimated as described previously (4), and the ranked diffusivities D₁, D₂, D₃, their associated mean diffusivity (MD) and fractional anisotropy (FA), and transmural span of the helix angle (HA) were averaged over manually segmented left ventricular regions. To elucidate the source of the DTI measurements, bi-compartmental DTI experiments (5), consisting of 48 gradients directions and 12 b-values ranging from 100-5000 s/mm² were conducted in 5 hearts randomly selected from each 130 d and 5 mo groups. The scalar diffusivities for each compartment, MD and FA in addition to the angular difference (Δα ) between the principal eigenvectors (EV1) of the fast and slow compartments were obtained. Moreover, hearts from the same groups were cryo-sectioned, stained with wheat germ agglutinin (WGA) and DAPI and were imaged using a Leica SP8 confocal microscope, to highlight the extracellular space (ECS) and cell nuclei, respectively (3). Nucleus density (per mm²), myocyte size (length and width), and myocyte volume fraction (VF) were quantified in regions from 3 different transmural biopsies per heart. Bonferroni-corrected ANOVA and unpaired t-tests were performed for statistical analysis of the conventional and bi-compartmental DTI results, respectively, with P < 0.05 considered statistically significant. Maturation was associated with progressively decreased FA and increased MD, whereas HA appears qualitatively similar among the groups (Figure 1). Changes in scalar DTI parameters were linked to preferential increases of the radial diffusivities D₂ and D₃ (Table 1). The HA transmural span was not statistically different among the groups. Representative FA maps of bi-compartmental DTI (Fig. 2) show that maturation leads to lower FA in both fast and slow compartments. Moreover, bi-compartment DTI parameters summarized in Table 2 revealed that 5 mo group exhibited higher transverse diffusivities accompanied by lower FA in both compartments, higher VF of the fast compartment, and higher Δα between compartments. Analyses of confocal images (Fig. 2) from 3 sections in each heart revealed that hearts in the 5 mo group had larger myocytes (105±7 µm length and 18±2 µm width, vs. 46±5 µm length and 6±1 µm width), lower nucleus density (1802±209 vs. 6122±294 per mm² ), and higher intracellular VF (78±3% vs. 67±5%). The results indicate that tissue remodeling during development manifests in progressively increased DTI transverse diffusivities, decreased FA, and unchanged fiber orientation. Bi-compartmental DTI analysis reveals that the changes are mediated by similar changes of diffusivities in the compartments. The VF of the fast diffusion compartment numerically matches the intracellular VF from confocal microscopy, in both the magnitude and behavior. Because myocyte growth results in higher transverse diffusivities but unchanged longitudinal diffusivities in both compartments suggests that restriction due to physical compartmentalization may not be the predominant mechanism underlying myocardial DTI measurements. Nevertheless, the findings show that DTI is sensitive, and can potentially be used to noninvasively characterize microstructural remodeling of the myocardium during development. Furthermore, the data suggests a role for DTI to characterize cardiac remodeling in developing hearts affected by persistent pulmonary hypertension, which is a common condition in preterm births (2).