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.<\/p>\n
TBX18 revisited<\/p>\n
For more than a decade, TBX18 has been reported to reprogram working ventricular cardiomyocytes into sinoatrial\u2011node\u2013like pacemaker cells, based on widely cited high\u2011impact 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\u00a0high\u2011level TBX18 overexpression is profoundly toxic, causing severe myocardial fibrosis and scarring in mouse hearts, while the control did not.<\/p>\n
We found that supraphysiological TBX18 expression is\u00a0highly toxic for cardiomyocytes. That toxicity alone already questions the feasibility of TBX18 as a\u00a0clinically relevant biological pacemaker strategy.”<\/p>\n
Gerard Boink, senior author, cardiologist and principal\u00a0investigator, Amsterdam UMC<\/p>\n
Safe TBX18 levels, no pacemaker<\/p>\n
To separate toxicity from biological functionality, the researchers engineered an optimized AAV\u00a0cassette 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\u00a0repression of well-known targets such as Gja1 (Connexin43). Despite this controlled, non-toxic\u00a0expression, 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\u00a0key pacemaker gene programs, nor did it induced Hcn4 protein, or the pacemaker current If. “These\u00a0results give good reason to stop TBX18-based gene therapy<\/a> efforts in the heart,” says Boink. “Our\u00a0data show that even at realistic, non-toxic expression levels, you do not get pacemaker activity.\u00a0Instead of providing a therapy, you risk arrhythmia by ion channel dysregulation and electrical\u00a0instability.”<\/p>\n Vector artefacts exposed<\/p>\n 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<\/a> and scarring as the true driver of the earlier reported “TBX18 pacing” signals. The AAV system used in the present study avoided these confounders.\u00a0“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.\u00a0“Ironically, this dogma is founded on our own 20-year-old studies, which originally uncovered the role of TBX18 in sinus node development.”<\/p>\n Hcn2 proves its value<\/p>\n In contrast to TBX18, AAV\u2011mediated expression of the pacemaker channel Hcn2 produced robust, autonomically responsive ventricular pacing in the same rat complete AV\u2011block model. Co\u2011expression\u00a0of TBX18 did not improve Hcn2\u2011based 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\u2011therapy\u2013based pacemakers for patients with congenital complete heart block, and other pacemaker indications, where we are now actively moving forward.”<\/p>\n Valorization at Amsterdam UMC<\/p>\n Next to his work as cardiologist and group leader, Boink is also Chief Valorization Officer\u00a0 for Amsterdam Cardiovascular Sciences and Chief Scientific Officer at PacingCure. “I feel\u00a0 privileged to work in an environment where valorization and real\u2011world impact are just as\u00a0 important as publishing in high impact journals, which obviously also has our priority,” says\u00a0Boink.<\/p>\n Source:<\/p>\n Amsterdam University Medical Center<\/a><\/p>\n Journal reference:<\/p>\n