AAV-mediated long-term TBX18 expression causes cardiac fibrosis and fails to induce pacemaker activity in rodents
Jianan Wang, Mathilde R. Rivaud, Mischa Klerk, Arie R. Boender, Ruud N. Visser, Rinske Sparrius, Hee Young Lee, Karel van Duijvenboden, Huiling Zhou, Yuting Yang, Emiel J.M. Kramer, Kyung Ho Park, Larry C. Park, Silke Schrödel, Christian Thirion, Eric Ehrke-Schulz, Anja Ehrhardt, Osne F. Kirzner, Klaus Neef, Hanno L. Tan, Arie O. Verkerk, Vincent M. Christoffels, Gerard J.J. Boink
Jianan Wang, Mathilde R. Rivaud, Mischa Klerk, Arie R. Boender, Ruud N. Visser, Rinske Sparrius, Hee Young Lee, Karel van Duijvenboden, Huiling Zhou, Yuting Yang, Emiel J.M. Kramer, Kyung Ho Park, Larry C. Park, Silke Schrödel, Christian Thirion, Eric Ehrke-Schulz, Anja Ehrhardt, Osne F. Kirzner, Klaus Neef, Hanno L. Tan, Arie O. Verkerk, Vincent M. Christoffels, Gerard J.J. Boink
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Research Article Cardiology Development

AAV-mediated long-term TBX18 expression causes cardiac fibrosis and fails to induce pacemaker activity in rodents

Abstract

Gene therapy–based biological pacemakers have been proposed as an alternative to their hardware-based counterparts. In this context, short-term ectopic expression of the T-box transcription factor 18 (TBX18) in the ventricle has been reported to generate potent, short-term pacemaker function in various animal models. Here, we investigated the impact of adeno-associated virus–mediated (AAV-mediated), long-term expression of TBX18 and compared the outcomes with those of the pacemaker ion channel hyperpolarization-activated cyclic nucleotide-gated potassium and sodium channel 2 (Hcn2). Our findings revealed that CMV-driven ectopic TBX18 expression in mouse hearts led to severe cardiac fibrosis. At lower, nonfibrogenic levels, TBX18 maintained its transcriptional function but failed to induce pacemaker phenotypes. TBX18-expressing cells showed suppressed expression of key working myocardial genes, but the pacemaker gene program was not induced. Electrophysiological studies showed abnormal automaticity in TBX18-expressing cells, combined with prolonged repolarization and various current changes. However, no hyperpolarization-activated funny current was detected. In a complete atrioventricular block rat model, AAV-mediated Hcn2 expression induced robust ectopic pacemaker activity in the presence of isoproterenol, whereas TBX18 expression neither generated such activity nor augmented Hcn2-mediated pacing. In conclusion, at functionally nonfibrogenic levels, TBX18 is neither sufficient nor necessary to induce pacemaker activity. In contrast, Hcn2 generates reliable pacing, making it a more viable candidate for biological pacemaker development.

Authors

Jianan Wang, Mathilde R. Rivaud, Mischa Klerk, Arie R. Boender, Ruud N. Visser, Rinske Sparrius, Hee Young Lee, Karel van Duijvenboden, Huiling Zhou, Yuting Yang, Emiel J.M. Kramer, Kyung Ho Park, Larry C. Park, Silke Schrödel, Christian Thirion, Eric Ehrke-Schulz, Anja Ehrhardt, Osne F. Kirzner, Klaus Neef, Hanno L. Tan, Arie O. Verkerk, Vincent M. Christoffels, Gerard J.J. Boink

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Figure 3

Dose-titrated expression cassette achieves low yet functional TBX18 expression.

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Dose-titrated expression cassette achieves low yet functional TBX18 expr...
(A) Schematic representation of expression cassette with and without upstream open reading frame (uORF). Translation start sites are made bold. (B) Direct fluorescence images of HEK293T (HEK) cells transfected with GFP and uORF-GFP plasmids. Yellow arrows indicate the GFP-positive cells. (C) Percentage of GFP-positive cells and (D) mean fluorescence intensity of HEK cells transfected with GFP or uORF-GFP plasmids (n = 3). (E) Example Western blot and (F) quantification of TBX18 protein expression in HEK cells transfected with TBX18 or uORF-TBX18 plasmids (n = 3). (G) AAV vectors used for transduction and Western blot of TBX18 protein expression in transduced cardiomyocytes. cTnT, cardiac troponin T; NC, negative control. (H) Quantification of TBX18 protein expression in cardiomyocytes transduced with various TBX18 vectors (n = 3). (I) Expression of pacemaker genes and (J) working myocardial genes in cardiomyocytes transduced with various TBX18 vectors (n = 6) and 1 GFP vector (n = 5). Data are presented as the mean ± SEM. Data were compared using Mann-Whitney U test (H), 1-way ANOVA with post hoc Holm-Šídák test (C, D, and F), or 2-way ANOVA with post hoc Holm-Šídák test (I and J). *P < 0.05 and **P < 0.01.