MYL4 Identifies Intramural Anatomy of Purkinje Fibers in Human Hearts
Matthew L. Hillestad, Matthew Amontree, Ryan Mahlberg, Monique S Bagwell, Skylar A. Rizzo, D. Kent Arrell, Parisa K Kargaran, Ruben Crespo‐Diaz, Faisal F. Syed, Elli N Lockhart, Tyra A. Witt, Paul G. Stalboerger, André Terzic, Christopher J. McLeod, Christopher V. DeSimone, Samuel J. Asirvatham, Atta Behfar
Abstract
BACKGROUND: Cardiac Purkinje fibers (PFs) orchestrate myocardial synchrony, but in regions of myocardial scarring, drive ventricular arrhythmia. It is hypothesized that arrhythmias refractory to ablation may be driven by PFs deep in the scarred myocardium. However, knowledge of human PF anatomy remains reliant on data generated in animal models, obscuring understanding of the substrate underpinning dysrhythmia and pacing strategies. OBJECTIVES: This study sought to establish and utilize a human biomarker to delineate PF anatomy in humans. METHODS: RNA-sequencing and differential expression analysis of transcriptomes from human hearts (N = 10) revealed 99 genes up-regulated in PFs vs ventricular myocardium and endocardium. Whole-mount megablock cross-sectional analysis was performed with cardiac troponin, MYL4, periodic acid-Schiff, Masson's Trichrome, pro-ANP, and GJA5 colocalization. RESULTS: Comparative transcriptomics from human hearts (N = 10) identified myosin light chain 4 (MYL4) as a promising human PF marker for distinguishing conductive from contractile myocardium. Using PF-rich bundle branch and subendocardial regions, MYL4 specificity was validated using canonical PF markers in human (n = 3), dog (n = 3), goat (n = 3), and pig (n = 3) hearts. Cross-sectional histology of the entire human ventricular myocardium uncovered a deep-seated network of transmurally intercalated, MYL4-positive PFs. This previously unrecognized distribution of the cardiac conduction system accounted for over 60% of human myocardial PF content. CONCLUSIONS: Such significant intramural prevalence exposes limitations associated with treatment approaches based on dogma that PF anatomy is restricted to the subendocardium. MYL4 here enabled a comprehensive visualization of human cardiac conduction system, offering an intricate bioanatomical map for heart rhythm management.