Oct. 21, 2025
Mayo Clinic cardiovascular researchers uncovered a new intramyocardial Purkinje tissue finding. The novel biomarker myosin light chain 4 (MYL4) was identified through RNA sequencing. It is described for the first time in a study published in JACC: Clinical Electrophysiology.
"The study identified novel biomarkers specific to the human Purkinje network. With this molecular blueprint, we were able to visualize a new anatomy for the cardiac conduction system," says Atta Behfar, M.D., Ph.D., a cardiologist at Mayo Clinic in Rochester, Minnesota, and senior author of the study. "This is the first time that intramural Purkinje fibers (PFs) architecture has been comprehensively described in humans."
Key discovery
The cardiac PFs are a subpopulation of cardiomyocytes (CMs) and terminal part of the cardiac conduction system. PFs drive contraction of a beating heart by relaying electrical impulses from upstream cardiac conduction system components to a larger cohort of working CMs.
"This study presents a key discovery: A deep-seated network of transmurally intercalated, MYL4-positive PFs within the human ventricular myocardium, which accounts for over 60% of the total myocardial PF content. Traditionally, PFs have been understood as being primarily restricted to the subendocardial layer (the inner surface of the heart muscle), based largely on animal models and historic human histological data," says Dr. Behfar. "However, using advanced transcriptomics and immunostaining with the novel biomarker MYL4, we were able to visualize and quantify an extensive intramural PF network that extends deep into the myocardium, intercalating with working cardiomyocytes."
With the reliance on animal analogies and nonspecific markers in prior studies, the depth and complexity of human PF distribution was underestimated. "The finding was enabled by whole-mount megablock cross-sectional analysis of human hearts," says Dr. Behfar. "The study originated from comparative transcriptomics on 10 human hearts, identifying 99 upregulated genes in PF-rich regions, with MYL4 emerging as a top candidate for its specificity to conductive myocardium."
The study is transformative for both cardiac electrophysiology and arrhythmia research. "It challenges long-standing anatomical dogma and may help guide both ablative and pacing strategies in our patient population," says Dr. Behfar.
Meeting critical needs
The idea stemmed from a critical gap in human cardiac anatomy: The lack of a specific, consistent biomarker for PFs in humans.
"Purkinje fibers are essential for rapid ventricular conduction and are implicated in arrhythmias, but their precise anatomy in humans remained poorly mapped due to nonspecific markers that could not distinguish PFs from other cell types accurately. This ambiguity hindered progress in understanding arrhythmia mechanisms, especially in scarred or ischemic myocardium," says Dr. Behfar. "The need arose from clinical challenges in electrophysiology, such as arrhythmias refractory to catheter ablation and suboptimal outcomes in cardiac resynchronization therapy. We hypothesized that deeper, unrecognized PF networks could be involved, necessitating a human-specific biomarker."
Rethinking interventions
The findings underscore the need to rethink PF-related pathologies and interventions. "This study provides a new bioanatomical map of the cardiac conduction system, enhancing understanding of myocardial synchrony, arrhythmia substrates in scarred tissue and potential pacing strategies. Overall, it shifts the paradigm from a surface-only view of PFs to a three-dimensional, intramural network, opening avenues for more-precise diagnostics and therapies in heart rhythm disorders," says Dr. Behfar.
A clinical focus on 3D strategies improves outcomes in arrhythmia management and reduces morbidity in patients with structural heart disease.
"The unexpected revelation was how MYL4 staining unveiled a transmurally intercalated system, suggesting PFs are more integrated with working myocardium than anticipated," says Dr. Behfar. "This implies a more resilient conduction network but also a hidden vulnerability to arrhythmias."
Translational potential
This research has direct translational potential for clinical electrophysiology:
Improved arrhythmia ablation. Revealing intramural PFs as potential substrates for refractory ventricular arrhythmias could guide deeper, more-precise catheter ablations or cryoablation techniques, reducing recurrence rates and procedural risks.
Enhanced cardiac pacing and resynchronization. Optimizing lead placement in pacemakers or CRT devices, with a better anatomical map targeting intramural PFs for more synchronous ventricular activation, especially in patients with heart failure.
Therapeutic innovation. Understanding intramural PFs may inform gene therapies or pharmacological targeting to modulate conduction in ischemic or fibrotic hearts, potentially preventing sudden cardiac death.
Next steps
This methodological innovation of combining RNA sequencing with immunostaining offers a reproducible approach for future PF studies.
The study shows the need for human-centric data since animal PF anatomy varies significantly. Future advancements could include functional validation, larger cohort studies and therapeutic trials. There's also a possibility for developing noninvasive tools to visualize intramural PFs in vivo, enabling clinical trials for arrhythmia prediction.
And beyond imaging integration, there could be cross-disciplinary collaboration. "This may include partnering with bioengineers for 3D PF models or AI-driven anatomical simulations to predict arrhythmia risks," says Dr. Behfar. "These advancements could rapidly translate to personalized heart rhythm therapies in the future."
For more information
Hillestad ML, et al. MYL4 identifies intramural anatomy of Purkinje fibers in human hearts. JACC: Clinical Electrophysiology. 2025;11:1718.
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