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Muscarinic Receptors Promote Pacemaker Fate at the Expense of Secondary Conduction System Tissue in Zebrafish


Muscarinic Receptors Promote Pacemaker Fate at the Expense of Secondary Conduction System Tissue in Zebrafish

Martina S Burczyk et al. JCI Insight.


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.

Keywords: Arrhythmias; Cardiology; Development; Embryonic development; G-protein coupled receptors.

Conflict of interest statement

Conflict of interest: The lab of MP has received a research grant from the Boehringer Ingelheim Ulm University BioCenter, which is a research program that is cofunded by the Ministry of Research of the State of Baden-Wuerttemberg, Boehringer Ingelheim, and the Medical School at Ulm University.


Figure 1
Figure 1. Compound screen reveals 9 drugs causing cardiac arrhythmia in zebrafish.
Zebrafish embryos were cultured in 50 μM compound in egg water or in egg water containing 1% DMSO from tailbud stage on. At the 48 hpf stage, embryos were individually and manually assessed for arrhythmia (A), formation of pericardiac or inflow tract edema (B), and atrial heart rates (C). (A) Eight out of the 90 drugs of the LOPAC Pfizer library produced arrhythmia in zebrafish embryos. DMSO-treated embryos never showed arrhythmia. n = 4–7 treatments with 31–56 embryos. (B) Seven drugs impaired overall heart function, resulting in edema formation. Dashed line indicates the percentage of embryos developing edema under DMSO treatment. n = 4–7 treatments with 25–83 embryos. (C) Tolterodine-treated embryos showed the highest variability in heart rate (shown as bpm) compared with DMSO-treated embryos (dashed line). Circles display heart rates of individual embryos. n = 10–158 embryos. Bar graphs display mean ± SEM, with circles indicating individual experiments. (A and B) Data analyzed by 1-way ANOVA, with *P < 0.05, **P < 0.01, and ****P < 0.0001.
Figure 2
Figure 2. Cardiac performance and conduction system function are compromised by increasing concentrations of tolterodine.
(A) Zebrafish embryos were treated from tailbud stage on with increasing concentrations of tolterodine. At 48 hpf, embryos were assessed for atrial arrhythmia. n = 3–11 experiments with 74–137 embryos. (B) Tolterodine-treated embryos developed atrioventricular conduction blocks (AV-blocks). A value of 50% corresponds to a 2:1 AV-block with the atrium beating twice as fast as the ventricle; 100% AV-block indicates a silent ventricle. n = 45–56 embryos. (C) Zebrafish were also assessed for edema formation as a general readout for impaired cardiac function. n = 4 experiments with 73–85 embryos. (D) Live images of 48 hpf embryos after treatment with increasing concentrations of tolterodine. Arrows indicate pericardiac edema and arrowheads inflow tract edema. Scale bar: 200 μm. All graphs display mean ± SEM.
Figure 3
Figure 3. Tolterodine affects heart function in Xenopus in a similar fashion as in zebrafish.
Xenopus embryos were treated from stage 12.5 until 42 in frog water and analyzed by hand. In BD numbers of animals used are indicated below the graphs. (A) Live images of stage 42 Xenopus embryos treated with vehicle (1% DMSO) or 50 μM tolterodine. Arrow indicates pericardiac effusion. (B) Tolterodine treatment results in pericardiac edema in Xenopus embryos. n = 8 experiments. ***P = 0.0002, 2-tailed Mann-Whitney U test. (C) A significant number of Xenopus embryos developed arrhythmia upon tolterodine treatment. n = 8 experiments. ***P = 0.0007, 2-tailed Mann-Whitney U test. (D) Tolterodine induced bradycardia in Xenopus embryos. n = 8 experiments. ****P < 0.0001, 2-tailed Mann-Whitney U test. Graphs show mean of individual experiments (B and C) or individual embryos (D). Red line, median.
Figure 4
Figure 4. Mice lacking both M2 and M3 muscarinic receptors show impaired cardiac performance.
(A) Representative echocardiograms of WT and M2/M3 double-KO mice reveal no abnormalities in cardiac rhythm. n = 4–5 mice per genotype (5–6 months of age, mixed sex). (B) Heart rate was not altered in M2/M3 double-KO mice. n = 4–5 mice per genotype. (C) M2/M3 double-KO mice had a higher heart weight (HW) to body weight (BW) ratio. This was due to an overall reduced size of M2/M3 double-KO mice. **P = 0.0057, 2-tailed t test. n = 4–5 mice per genotype. (D) M2/M3 double-KO mice had a reduced ejection fraction (EF). **P = 0.0040, 2-tailed t test. n = 4–5 mice per genotype. (E) Fractional shortening (FS) was also reduced in M2/M3 double-KO mice. *P = 0.0129, 2-tailed t test. n = 4–5 mice per genotype. (F) At the same time, left ventricular end-diastolic diameter (LVED) was increased in M2/M3 double-KO mice. *P = 0.0272, 2-tailed t test. n = 4–5 mice per genotype. (G) M2/M3 double-KO mice further displayed a larger left ventricular end-diastolic volume (LVEV). *P = 0.0303, 2-tailed t test. n = 4–5 mice per genotype. Bar graphs show mean ± SEM and circles indicate individual mice.
Figure 5
Figure 5. Zebrafish treatment with antimuscarinic drugs favoring M2 over M3 receptors does not phenocopy the effects seen with tolterodine.
(A) Live images of 48 hpf embryos after treatment with vehicle (DMSO) or 50 μM himbacine (himba.) or AF-DX 116 (AF-DX.). Scale bar: 200 μm. (B) Himbacine treatment did not result in arrhythmia. n = 3 experiments with 63–65 embryos. (C) Himbacine did not produce edema. n = 3 experiments with 63–65 embryos. (D) The heart rate was not changed upon himbacine treatment. n = 63–65 embryos. (E) AF-DX116 treatment did not promote arrhythmia development. n = 3 experiments with 62–67 embryos. (F) Edema did not develop upon AF-DX 116 treatment. n = 3 experiments with 62–67 embryos. (G) Heart rates were not changed when treated with AF-DX116. n = 62–67 embryos. Circles indicate individual experiments (B, C, E, and F) or embryos (D and G); median is shown with red line. All data analyzed by 2-tailed Mann-Whitney U tests.
Figure 6
Figure 6. Antimuscarinic compounds favoring M3 over M2 receptors phenocopy tolterodine treatment in zebrafish embryos.
(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.
Figure 7
Figure 7. Tolterodine treatment causes elongation of the AVC.
(A) Whole-mount live images of fluorescent zebrafish hearts [Tg(myl7:EGFP)] at 48 hpf. The AVC (indicated by a red bracket) is elongated upon tolterodine treatment. (B) Cardiomyocyte number remained unaltered in tolterodine-treated zebrafish. Images show confocal stacks of Tg(-5.1myl7:DsRed2-NLS) zebrafish. DsRed-expressing nuclei of cardiomyocytes were enhanced by staining with an anti-DsRed antibody. Atria were visualized by staining of atrial myosin (S46). Graph shows individual embryos as circles and mean ± SEM. n = 7–10 embryos. (C) Tolterodine did not induce apoptosis in cardiomyocytes as shown by the lack of cleaved caspase-3–positive cardiomyocytes. Images show confocal stacks of Tg(myl7:EGFP) hearts. Circles show individual embryos. P = 0.1429, 2-tailed Mann-Whitney U test. n = 5 embryos each. (D) Chamber specification was not affected by tolterodine. Atrial (amhc) and ventricular (vmhc) myosin visualized by double in situ hybridization. n = 3 experiments with 34–50 embryos. Scale bars: 100 μm (A), 50 μm (BD).
Figure 8
Figure 8. Tolterodine promotes pacemaker cell fate at the expense of AVC cells.
(AD) Representative images of whole-mount in situ hybridizations at 48 hpf. A, atrium; V, ventricle. Numbers of embryos studied are indicated below the bars. n = 3–4 experiments. (A) AVC cells aberrantly express anf, which is characteristic of working myocardium and normally excluded from the AVC. **P = 0.0013, 2-tailed t test with Welch’s correction. (B) Upon tolterodine treatment, notch1b expression was lost. P = 0.100, 2-tailed Mann-Whitney U test. (C) Tolterodine provoked an increase in the pacemaker marker fgf13a. ***P = 0.0005, 2-tailed t test with Welch’s correction. (D) Tolterodine increased tbx18 expression in the area of the inflow tract. *P = 0.0158, 2-tailed t test with Welch’s correction. (E) Quantitative PCR (qPCR) analysis of isolated hearts (bright-field images) showed the induction of a pacemaker program by tolterodine. n = 3–6 experiments. *P = 0.0142 (hcn4), 0.0193 (cx43), and 0.0213 (tbx18), paired t test. Scale bars: 50 μm. Bar graphs show mean ± SEM (except for B and tbx18 in E: red line is median). Circles show individual experiments. Arrows indicate changes in expression between panels.
Figure 9
Figure 9. Treatment during CPC stages is sufficient to affect conduction system development.
(A) Tolterodine treatment induced the expression of genes characteristic for pacemaker cells (Hcn4, Shox2) in cardiomyocytes differentiated from human iPSCs. Graph shows qPCR data of treatments. Line indicates mean; box shows minimum and maximum. (B) In zebrafish embryos, the muscarinic receptor M3a was increasingly expressed during CPC differentiation stages. qPCRs of zebrafish at the indicated stages. Chrm3a normalized to gapdh. Red line, median; ss, somite stages. (C) Schematic illustration of different treatment regimens in zebrafish embryos producing AVC elongation in more than 50% of the embryos. (D) Tolterodine treatment between tailbud and 20 ss is sufficient to induce anf expression in the presumptive AVC. Scale bar: 50 μm. Bar graph depicts the number of embryos with or without anf expression in the AVC. n = 2 experiments. The number of individual embryos is given in the graph. ****P < 0.0001, 2-sided Fisher’s exact test. (E) Tolterodine treatment during heart progenitor specification and differentiation stages results in elevated isl1 expression that reaches into the atrium. Scale bar: 50 μm. Bar graph summarizes 2 experiments (embryo number indicated in the graph itself). ****P < 0.0001, 2-sided Fisher’s exact test. SAN, sinoatrial node.

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