Muscarinic receptors promote pacemaker fate at the expense of secondary conduction system tissue in zebrafish
Martina S. Burczyk, Martin D. Burkhalter, Teresa Casar Tena, Laurel A. Grisanti, Michael Kauk, Sabrina Matysik, Cornelia Donow, Monika Kustermann, Melanie Rothe, Yinghong Cui, Farah Raad, Svenja Laue, Allessandra Moretti, Wolfram-H. Zimmermann, Jürgen Wess, Michael Kühl, Carsten Hoffmann, Douglas G. Tilley, Melanie Philipp
Martina S. Burczyk, Martin D. Burkhalter, Teresa Casar Tena, Laurel A. Grisanti, Michael Kauk, Sabrina Matysik, Cornelia Donow, Monika Kustermann, Melanie Rothe, Yinghong Cui, Farah Raad, Svenja Laue, Allessandra Moretti, Wolfram-H. Zimmermann, Jürgen Wess, Michael Kühl, Carsten Hoffmann, Douglas G. Tilley, Melanie Philipp
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Research Article Cardiology Development

Muscarinic receptors promote pacemaker fate at the expense of secondary conduction system tissue in zebrafish

Abstract

Deterioration or inborn malformations of the cardiac conduction system (CCS) interfere with proper impulse propagation in the heart and may lead to sudden cardiac death or heart failure. Patients afflicted with arrhythmia depend on antiarrhythmic medication or invasive therapy, such as pacemaker implantation. An ideal way to treat these patients would be CCS tissue restoration. This, however, requires precise knowledge regarding the molecular mechanisms underlying CCS development. Here, we aimed to identify regulators of CCS development. We performed a compound screen in zebrafish embryos and identified tolterodine, a muscarinic receptor antagonist, as a modifier of CCS development. Tolterodine provoked a lower heart rate, pericardiac edema, and arrhythmia. Blockade of muscarinic M3, but not M2, receptors induced transcriptional changes leading to amplification of sinoatrial cells and loss of atrioventricular identity. Transcriptome data from an engineered human heart muscle model provided additional evidence for the contribution of muscarinic M3 receptors during cardiac progenitor specification and differentiation. Taken together, we found that muscarinic M3 receptors control the CCS already before the heart becomes innervated. Our data indicate that muscarinic receptors maintain a delicate balance between the developing sinoatrial node and the atrioventricular canal, which is probably required to prevent the development of arrhythmia.

Authors

Martina S. Burczyk, Martin D. Burkhalter, Teresa Casar Tena, Laurel A. Grisanti, Michael Kauk, Sabrina Matysik, Cornelia Donow, Monika Kustermann, Melanie Rothe, Yinghong Cui, Farah Raad, Svenja Laue, Allessandra Moretti, Wolfram-H. Zimmermann, Jürgen Wess, Michael Kühl, Carsten Hoffmann, Douglas G. Tilley, Melanie Philipp

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Figure 6

Antimuscarinic compounds favoring M3 over M2 receptors phenocopy tolterodine treatment in zebrafish embryos.

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Antimuscarinic compounds favoring M3 over M2 receptors phenocopy toltero...
(A) Live images of 48 hpf embryos after treatment with vehicle (DMSO) or 50 μM solifenacin or zamifenacin. Arrow indicates the edema. Scale bar: 200 μm. (B) Solifenacin treatment caused dose-dependent arrhythmia. n = 3–8 experiments with 85–165 embryos. (C) Zebrafish treated with increasing concentrations of solifenacin developed AV-blocks. n = 3–8 experiments with 85–165 embryos. (D) Solifenacin caused formation of edema with increasing concentrations. n = 3–8 experiments with 85–165 embryos. (E) Zebrafish embryos treated with zamifenacin developed arrhythmia at low micromolar concentrations. At higher concentrations, both chambers became silent. n = 3–6 experiments with 59–144 embryos. (F) Zamifenacin treatment provoked AV-blocks. n = 3–6 experiments with 59–144 embryos. (G) Edema formation occurred in a dose-dependent manner upon zamifenacin treatment. n = 3–6 experiments with 59–103 embryos. Graphs display mean ± SEM.