Histology-validated quantitative MRI characterization of collagen-rich Purkinje fiber-associated pathways in the ex vivo porcine heart

Abstract Objective Collagen-rich Purkinje fiber-associated (PF-associated) pathways within the subendocardial layer are small, compositionally distinct structures that are challenging to characterize noninvasively. This study aimed to evaluate whether quantitative magnetic resonance imaging (qMRI) can characterize collagen-rich PF-associated pathways using histology as a structural ground truth. Materials and methods Ex vivo porcine left ventricular specimens were imaged at 3 T using relaxation-based mapping techniques, including T RAFF , T 1ρ , T 2 and T 1 mapping. Corresponding Masson’s trichrome sections were processed into optical density maps to segment myocardium, collagen and PF-associated regions. Histology-defined regions of interest were used to identify collagen-enriched PF-associated pathways and remote myocardium. Relaxation time contrast was quantified using relative relaxation time difference (RRTD). Statistical comparisons were performed using paired t -tests with Benjamini–Hochberg correction. Results Histology confirmed collagen-enriched PF-associated pathways within subendocardial tissue and false tendons. Optical density-based segmentation reliably differentiated tissue components with low inter-section variability. Collagen-rich PF-associated regions showed significantly higher T RAFF , T 1ρ , T 2 , and T 1 values compared with remote myocardium. RRTD analysis indicated that T RAFF and T 1ρ provided the strongest relative contrast among the evaluated parameters. Conclusion Histology-validated qMRI enables characterization of collagen-rich PF-associated pathways in the ex vivo porcine heart. These findings demonstrate the sensitivity of relaxation-based MRI techniques to collagen-rich PF-associated pathways and support further investigation of cardiac conduction system imaging. Key points Question Noninvasive MRI characterization of collagen-rich PF-associated pathways remains challenging because these small conduction structures are difficult to identify. Findings Histology-referenced MRI differentiated collagen-rich PF-associated pathways from those of the remote myocardium, with rotating-frame relaxation methods providing the strongest contrast. Relevance statement qMRI relaxation mapping enables histology-validated characterization of collagen-rich PF-associated pathways, supporting the development of noninvasive imaging approaches for studying cardiac conduction system microstructure.