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 1

Injection of AdV vectors leads to ectopic pacing in a complete AV-block rat model.

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Injection of AdV vectors leads to ectopic pacing in a complete AV-block ...
(A) An example ECG tracing showing complete AV-block in rats after AV node ablation. P wave and QRS complex are indicated on top. (B) Experimental design. (C) Immunofluorescence staining images of GFP and TBX18 in rat hearts injected with AdV-GFP and AdV-TBX18, respectively. Scale bar: 50 μm. (D) Example ECG tracings of rats injected with saline, AdV-GFP, and AdV-TBX18, before and after the administration of isoproterenol. (E) Percentage of ectopic pacing and (F) average heart rate in rats injected with saline (n = 4), AdV-GFP (n = 6), and AdV-TBX18 (n = 6). (G) Picrosirius red staining images and (H) fibrosis quantification of rat hearts injected with saline (n = 4), AdV-GFP (n = 6), and AdV-TBX18 (n = 6). Scale bar: 300 μm. Data are shown as the mean ± SEM. Data were compared using 2-way ANOVA (E and F) or 1-way ANOVA (H) with a post hoc Holm-Šídák test. *P < 0.05, **P < 0.01, and ***P < 0.001.