Researchers at Amsterdam UMC have overturned an important assumption in the field of biological pacemakers. New preclinical studies show that the transcription factor TBX18 does not produce true biological pacemaker activity, but the ion channel Hcn2 produces potent pacemaker function in the heart.
Rethinking TBX18
Based on widely cited and influential papers, TBX18 has been reported for more than a decade to reprogram active ventricular cardiomyocytes into sinoatrial node-like pacemaker cells. A team at Amsterdam UMC systematically revisited this claim using a low-immunogenic adeno-associated virus (AAV) vector and detailed electrophysiology. They showed for the first time that conventional high levels of TBX18 overexpression are highly toxic, causing severe myocardial fibrosis and scarring in mouse hearts, but not in controls.
We found that supraphysiological TBX18 expression is highly toxic to cardiomyocytes. Its toxicity alone already calls into question the feasibility of TBX18 as a clinically relevant biological pacemaker strategy. ”
Gerald Boink, lead author, cardiologist and principal investigator at UMC Amsterdam
Safe TBX18 level, no pacemaker
To separate toxicity and biological function, the researchers designed an optimized AAV cassette that reduced TBX18 protein levels to approximately 1% of expression by conventional CMV. This completely prevented fibrosis and further maintained the transcriptional activity of TBX18, effectively suppressing well-known targets such as Gja1 (Connexin43). Despite this controlled nontoxic expression, cardiomyocytes did not acquire a true pacemaker phenotype. TBX18 represses multiple agonist muscle cell genes, leading to abnormal action potentials. However, the critical pacemaker gene program was not induced, nor was the Hcn4 protein or the pacemaker current If. “These results provide good reason to halt TBX18-based gene therapy efforts in the heart,” Boink says. “Our data show that even at realistic, non-toxic expression levels, pacemaker activity is not achieved. Instead of providing therapy, we risk causing arrhythmias through ion channel dysregulation and electrical instability.”
Vector artifacts exposed
In a rat model of complete atrioventricular (AV) block, both adenovirus (AdV)-TBX18 and AdV control caused similar ectopic pacing and extensive focal fibrosis, pointing to AdV vector-associated inflammation and scarring as the true drivers of the previously reported “TBX18 pacing” signal. The AAV system used in this study avoided these confounding factors. “There is a tendency in some parts of the field to overestimate single, high-impact papers,” Boink said. “It may inadvertently elevate artificial concepts like TBX18-mediated reprogramming to the status of dogma. Ironically, this dogma is based on our own 20 years of research that originally revealed the role of TBX18 in sinus node development.”
Hcn2 proves its worth
In contrast to TBX18, AAV-mediated expression of the pacemaker channel Hcn2 produced robust and autonomically responsive ventricular pacing in the same rat complete AV block model. Coexpression of TBX18 did not improve Hcn2-based pacing, highlighting that TBX18 could not be used to further promote HCN-based pacing. “This study also shows that an efficient biological pacemaker can be created with Hcn2 alone,” Boink says. “This has direct implications for the development of gene therapy-based pacemakers for patients with congenital complete heart block and other pacemaker indications, which we are currently actively developing.”
Valuation at Amsterdam UMC
In addition to his work as a cardiologist and group leader, Mr. Boink is also Chief Value Officer of the Department of Cardiovascular Sciences Amsterdam and Chief Scientific Officer of Pacing Cure. “We are privileged to work in an environment where valuation and real-world impact are as important as publishing in high-impact journals, and that is clearly our priority as well,” says Boink.
sauce:
Amsterdam University Medical Center
Reference magazines:
Wang, J. others. (2026). AAV-mediated long-term expression of TBX18 causes cardiac fibrosis and fails to induce pacemaker activity in rodents. clinical research journal. DOI: 10.1172/jci190632. https://www.jci.org/articles/view/190632
