Amsterdam UMC researchers overturn long-standing biological pacemaker dogma

Researchers from Amsterdam UMC have overturned a key assumption in the biological pacemaker field. In a new preclinical study they show that the transcription factor TBX18 does not generate true biological pacemaker activity, while the ion channel Hcn2 does produce robust pacemaker function in the heart.

TBX18 revisited

For more than a decade, TBX18 has been reported to reprogram working ventricular cardiomyocytes into sinoatrial‑node–like pacemaker cells, based on widely cited high‑impact papers. Using low-immunogenic adeno-associated virus (AAV) vectors and detailed electrophysiology, the team from Amsterdam UMC systematically re-examined this claim. They first showed that conventional high‑level TBX18 overexpression is profoundly toxic, causing severe myocardial fibrosis and scarring in mouse hearts, while the control did not.

We found that supraphysiological TBX18 expression is highly toxic for cardiomyocytes. That toxicity alone already questions the feasibility of TBX18 as a clinically relevant biological pacemaker strategy."

Gerard Boink, senior author, cardiologist and principal investigator, Amsterdam UMC

Safe TBX18 levels, no pacemaker

To separate toxicity from biological functionality, the researchers engineered an optimized AAV cassette and reduced the TBX18 protein levels to about 1% of conventional CMV-driven expression. This completely prevented fibrosis, yet preserved transcriptional activity of TBX18 with effective repression of well-known targets such as Gja1 (Connexin43). Despite this controlled, non-toxic expression, cardiomyocytes did not acquire a genuine pacemaker phenotype. TBX18 suppressed multiple working-myocyte genes and lead to abnormal action potentials. However, it did not induce key pacemaker gene programs, nor did it induced Hcn4 protein, or the pacemaker current If. "These results give good reason to stop TBX18-based gene therapy efforts in the heart," says Boink. "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 by ion channel dysregulation and electrical instability."

Vector artefacts exposed

In a rat model of complete atrioventricular (AV) block, both adenoviral (AdV)-TBX18 and the AdV control produced similar ectopic pacing and extensive local fibrosis, pointing to AdV vector-related inflammation and scarring as the true driver of the earlier reported "TBX18 pacing" signals. The AAV system used in the present study avoided these confounders. "In some parts of the field, there has been a tendency to overvalue single high-impact papers," Boink notes. "That can unintentionally promote artefactual concepts, like TBX18-mediated reprogramming, to the status of a 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 proves its value

In contrast to TBX18, AAV‑mediated expression of the pacemaker channel Hcn2 produced robust, autonomically responsive ventricular pacing in the same rat complete AV‑block model. Co‑expression of TBX18 did not improve Hcn2‑based pacing, underscoring that TBX18 can also not be employed to further promote HCN-based pacing. "With this study we also show that an efficient biological pacemaker can be created with Hcn2 alone," says Boink. "That has direct implications for developing gene‑therapy–based pacemakers for patients with congenital complete heart block, and other pacemaker indications, where we are now actively moving forward."

Valorization at Amsterdam UMC

Next to his work as cardiologist and group leader, Boink is also Chief Valorization Officer  for Amsterdam Cardiovascular Sciences and Chief Scientific Officer at PacingCure. "I feel  privileged to work in an environment where valorization and real‑world impact are just as  important as publishing in high impact journals, which obviously also has our priority," says Boink.