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 6

Long-term TBX18 expression at noncytotoxic level leads to ionic remodeling of mouse cardiomyocytes.

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Long-term TBX18 expression at noncytotoxic level leads to ionic remodeli...
(A) Example action potentials (APs) recorded from TBX18-expressing and control cardiomyocytes (spontaneous/not stimulated). (B) Incidence of spontaneous activity. Number of cells with spontaneous activity and total number of cells studied are presented within the associated bar charts. (C) Example APs recorded from TBX18-expressing and control cardiomyocyte paced at 4 Hz. Inset: AP upstroke velocities. (D) Average AP parameters. RMP, resting membrane potential; APA, AP amplitude; APD20/50/90, AP durations at 20%/50%/90% repolarization; Vmax, maximal AP upstroke velocity (n = 8). (E) Example current traces at –100 and +50 mV of TBX18-expressing and control cardiomyocytes from a generic voltage-clamp. (F) Current-voltage (I-V) relationships of the quasi steady-state current. (G) Current densities of the inwardly rectifying K+ current (IK1) measured at –100 mV. (H) Current densities of the delayed rectifier outward K+ current (IK) measured at 50 mV. (I) I-V relationships of the hyperpolarization-activated funny current (If). (J) I-V relationships of Na+ current (INa). (K) Peak INa density measured at –40 mV. (L) Cell capacitance reflecting cell size. (F–L) n = 4 for Nluc and n = 5 for TBX18. Data are shown as the mean ± SEM. Data were compared using Fisher’s exact test (B) or Mann-Whitney U test (D, G, H, K, and L). *P < 0.05; **P < 0.01.