Amsterdam Scientists Overturn TBX18 Pacemaker Dogma in Landmark Preclinical Study
Researchers at Amsterdam UMC have dismantled a widely held assumption in cardiac biology, demonstrating that the transcription factor TBX18 does not generate genuine biological pacemaker activity — while identifying the ion channel Hcn2 as a robust and viable alternative. The findings, published in the Journal of Clinical Investigation, carry significant implications for gene therapy development in patients with heart block conditions.
A Decade of Assumptions Challenged
For more than ten years, TBX18 has been cited in high-impact literature as capable of reprogramming ventricular cardiomyocytes into sinoatrial-node–like pacemaker cells. The Amsterdam team, led by cardiologist and principal investigator Gerard Boink, subjected that claim to systematic scrutiny using low-immunogenic adeno-associated virus (AAV) vectors and detailed electrophysiology.
Their first finding was stark: conventional high-level TBX18 overexpression proved severely toxic, causing significant myocardial fibrosis and scarring in mouse hearts. Control subjects showed no such damage.
Safe Expression Levels Yield No Pacemaker Function
To disentangle toxicity from biological function, the researchers engineered an optimised AAV cassette that reduced TBX18 protein levels to approximately 1% of conventional CMV-driven expression. This eliminated fibrosis entirely while preserving transcriptional activity, including effective repression of known targets such as Gja1 (Connexin43).
Yet even at these controlled, non-toxic levels, cardiomyocytes failed to acquire a genuine pacemaker phenotype. TBX18 suppressed multiple working-myocyte genes and produced abnormal action potentials, but did not activate key pacemaker gene programmes, induce Hcn4 protein, or generate the pacemaker current If.
Boink was direct in his assessment: “Our data show that even at realistic, non-toxic expression levels, you do not get pacemaker activity. Instead of providing a therapy, you risk arrhythmia through ion channel dysregulation and electrical instability.”
Earlier Results Attributed to Vector Artefacts
The study also offers an explanation for previously reported positive results. In a rat model of complete atrioventricular (AV) block, both adenoviral (AdV)-TBX18 and an AdV control vector produced similar ectopic pacing and extensive local fibrosis. This points to AdV vector-related inflammation and scarring — rather than TBX18 itself — as the true driver of earlier pacing signals.
The AAV system used in the present study avoided these confounders, lending greater credibility to its negative findings on TBX18. Boink acknowledged the uncomfortable origins of the overturned dogma: “Ironically, this dogma is founded on our own 20-year-old studies, which originally uncovered the role of TBX18 in sinus node development.”
Hcn2 Emerges as the Credible Candidate
Where TBX18 failed, Hcn2 succeeded. AAV-mediated expression of the pacemaker channel produced robust, autonomically responsive ventricular pacing in the same rat AV-block model. Crucially, co-expression of TBX18 with Hcn2 offered no additional benefit, confirming that TBX18 cannot even serve as an adjunct to HCN-based approaches.
The implications for clinical development are considerable. Boink identified congenital complete heart block as a priority indication, noting that the team is “actively moving forward” on gene-therapy–based pacemaker development using Hcn2 alone.
Translational Ambitions
Boink holds dual roles as Chief Valorization Officer for Amsterdam Cardiovascular Sciences and Chief Scientific Officer at PacingCure, a company focused on translating these findings into clinical applications. The study underscores a broader methodological lesson for the field: the dangers of elevating single high-impact papers to the status of unquestioned dogma, particularly when underlying vector artefacts may have distorted early results.
The research is published as: Wang, J., et al. (2026). AAV-mediated long-term TBX18 expression causes cardiac fibrosis and fails to induce pacemaker activity in rodents. Journal of Clinical Investigation. DOI: 10.1172/jci190632.