MYL4 identifies intramural anatomy of Purkinje fibers in human hearts

ABSTRACTBACKGROUND: Cardiac Purkinje fibers (PFs) orchestrate myocardial synchrony but in regions of myocardial scarring driver 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: 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 upregulated in PFs versus ventricular myocardium and endocardium. Whole mount megablock cross sectional analysis was performed with cardiac troponin, MYL4, PAS, 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 seeded 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 subendocardium. MYL4 here enabled a comprehensive visualization of human cardiac conduction system, offering an intricate bioanatomical map for heart rhythm management.