Structural Characterization of Cardiac Free‐Running Purkinje Fibers Using Inhomogeneous Magnetization Transfer (ihMT): A Proof of Concept MRI‐Histology Approach

The cardiac Purkinje network plays a critical role in maintaining synchronized ventricular activation but remains difficult to image due to its fine and complex structure. Conventional MRI techniques lack sufficient contrast to distinguish the structural composition of Purkinje fibers (PFs). This study investigates the potential of inhomogeneous magnetization transfer (ihMT) as a novel contrast mechanism for visualizing and differentiating subregions of the Purkinje network. Five fixed ex vivo sheep hearts containing free-running PFs were scanned using a 9.4 T MRI system with a 2D ihMT RARE sequence. ihMTR maps were analyzed using manually defined regions of interest (ROIs) corresponding to free-running fibers, the Purkinje-myocardial junction (PMJ), and the surrounding myocardium. Histological analysis was performed on matched tissue sections to quantify collagen types I and III, adipocytes, Purkinje cells, and cardiomyocytes. Three ihMT protocols that produced high ihMTR values in free-running fibers (9.25-10.83%) and strong contrast relative to myocardium (2.00-2.17%) and the PMJ (2.99-3.40%) in 1 sample were selected and applied to all samples. Across all hearts, mean ihMTR values were consistently higher in free-running fibers compared to the PMJ (11.5 ± 1.5% vs 9.0 ± 2.9%). Histological analysis revealed significantly greater collagen content in free-running regions compared with the PMJ (72.4 ± 15.9% vs 31.1 ± 13.1%; p = 0.001), along with higher adipocyte content at the PMJ compared to free-running regions (12.3 ± 6.1% vs 3.8 ± 2.7%, not significant). Collagen type III was more prominent at the PMJ but remained a minor component overall. These findings demonstrate that ihMT imaging can distinguish PF subregions based on underlying microstructural differences, particularly collagen and adipocyte distribution. This study lays the groundwork for developing biophysical models to interpret ihMT signals in terms of tissue composition and microstructure, providing a foundation for future studies.