Yonsei Med J.  2018 Mar;59(2):279-286. 10.3349/ymj.2018.59.2.279.

The Role of Serotonin in Ventricular Repolarization in Pregnant Mice

• Cui S
• Park H
• Park H
• Mun D
• Lee SH
• Kim H
• Yun N
• Kim H
• Kim M
• Pak HN
• Lee MH
• Joung B

• KMID: 2418792
• DOI: http://doi.org/10.3349/ymj.2018.59.2.279

Abstract

PURPOSE
The mechanisms underlying repolarization abnormalities during pregnancy are not fully understood. Although maternal serotonin (5-hydroxytryptamine, 5-HT) production is an important determinant for normal fetal development in mice, its role in mothers remains unclear. We evaluated the role of serotonin in ventricular repolarization in mice hearts via 5Htr3 receptor (Htr3a) and investigated the mechanism of QT-prolongation during pregnancy.
MATERIALS AND METHODS
We measured current amplitudes and the expression levels of voltage-gated K⁺ (Kv) channels in freshly-isolated left ventricular myocytes from wild-type non-pregnant (WT-NP), late-pregnant (WT-LP), and non-pregnant Htr3a homozygous knockout mice (Htr3a(−/−)-NP).
RESULTS
During pregnancy, serotonin and tryptophan hydroxylase 1, a rate-limiting enzyme for the synthesis of serotonin, were markedly increased in hearts and serum. Serotonin increased Kv current densities concomitant with the shortening of the QT interval in WT-NP mice, but not in WT-LP and Htr3a(−/−)-NP mice. Ondansetron, an Htr3 antagonist, decreased Kv currents in WT-LP mice, but not in WT-NP mice. Kv4.3 directly interacted with Htr3a, and this binding was facilitated by serotonin. Serotonin increased the trafficking of Kv4.3 channels to the cellular membrane in WT-NP.
CONCLUSION
Serotonin increases repolarizing currents by augmenting Kv currents. Elevated serotonin levels during pregnancy counterbalance pregnancy-related QT prolongation by facilitating Htr3-mediated Kv currents.

Keyword

Serotonin receptor type 3; QT interval; pregnancy; voltage-gated K⁺ (Kv) current; membrane trafficking; serotonin

MeSH Terms

*Action Potentials/drug effects
Animals
Cell Membrane/drug effects/metabolism
Disease Models, Animal
Electrocardiography
Female
HSC70 Heat-Shock Proteins/metabolism
HSP90 Heat-Shock Proteins/metabolism
Heart Ventricles/drug effects/*metabolism
Mice, Inbred C57BL
Mice, Knockout
Myocytes, Cardiac/drug effects/metabolism
Potassium Channels/metabolism
Pregnancy
Rabbits
Rats, Sprague-Dawley
Receptors, Serotonin, 5-HT3/metabolism
Serotonin/*metabolism
Serotonin 5-HT3 Receptor Agonists/pharmacology
HSC70 Heat-Shock Proteins
HSP90 Heat-Shock Proteins
Potassium Channels
Receptors, Serotonin, 5-HT3
Serotonin 5-HT3 Receptor Agonists
Serotonin

Figure

• 

  Fig. 1 Serotonin, QTc and pregnancy. (A) Pregnancy increases serotonin and serotonin-related levels. Immunohistochemical staining (×40) of Htr3a in the ventricle of WT-NP, WT-LP, and Htr3a−/−-NP mice. (B) Comparison of Htr3a intensity among WT-NP, WT-LP, and Htr3a−/−-NP mouse hearts. (C) Effect of serotonin and m-CPBG on heart rate and QT and QTc intervals in WT-NP and WT-LP mice. (D) Effect of serotonin and m-CPBG on APD. Action potential tracings in WT-NP (left panels) and WT-LP (right panel) mice treated with serotonin, m-CPBG, and ondansetron plus serotonin. Comparison of APD90 in WT-NP and WT-LP mice. Error bars indicate means±SD. The number of cells is indicated next to the symbols. *p<0.05. WT-NP, wild-type non-pregnant; WT-LP, wild-type late-pregnant; Htr3a−/−, 5-hydroxytryptamine 3a-receptor knockout mice; Htr3a−/−-NP, non-pregnant Htr3a−/−; QTc, corrected QT; APD, action potential duration; APD90, action potential duration at 90%.

• 

  Fig. 2 Effects of serotonin and the Htr3 agonist on outward K+ currents in WT-NP and WT-LP mouse ventricular cardiomyocytes. (A) Outward K+ currents tracings from WT-NP (upper panels) and WT-LP (lower panels) ventricular cardiomyocytes treated with control buffer, serotonin, m-CPBG, and serotonin plus ondansetron. (B) Effects of serotonin, m-CPBG, and ondansetron on current-voltage (I–V) relationships for Kv current densities. (C) Effects of serotonin, m-CPBG, and serotonin plus ondansetron on Kv current densities at +60 mV in WT-NP and LP mice. The protocol is indicated in the inset, and the number of cells is indicated next to the symbols. *p<0.05, **p<0.001. Htr3a, 5-hydroxytryptamine receptor 3A; WT-NP, wild-type non-pregnant; WT-LP, wild-type late-pregnant.

• 

  Fig. 3 Enhanced Kv4.3 membrane trafficking in response to Htr3a-mediated serotonin stimulation in WT-NP, but not in WT-LP mice. (A) Co-immunoprecipitation of Kv4.3 and Htr3a in WT-NP and WT-LP mouse ventricular myocytes. (B) Co-immunoprecipitation illustrating enhanced co-precipitation of Htr3a and Kv4.3 following serotonin stimulation in WT-NP, but not in WT-LP mice. (C) Kv4.3 associated Htr3a level. (D) Immunostaining of Kv4.3 and Htr3a in WT-NP and WT-LP mice. Ventricular myocytes containing Kv4.3 (green) and Htr3a (red); yellow indicates co-localization. The lower panels illustrate the OD along the white bar across the cells. Images are representative of at least 8–10 experiments. Scale bar, 20 µm. (E) The comparison of the plasma membrane/endoplasmic reticulum OD ratio in WT-NP and WT-LP mice. Bar graphs illustrate mean±SD. *p<0.001. Htr3a, 5-hydroxytryptamine 3a-receptor; WT-NP, wild-type non-pregnant; WT-LP, wild-type late-pregnant; OD, optical density; NS, not significant.

• 

  Fig. 4 Serotonin improves Kv4.3 membrane trafficking through interaction with Hsc70 in WT-NP, but not in WT-LP mice. (A) Co-immunoprecipitation of Hsc70 with Kv4.3 illustrating enhanced co-precipitation of chaperones with Kv4.3 following serotonin stimulation (100 µmol/L for 10 min) in WT-NP, but not in WT-LP mice. (B) Kv4.3-associated Hsc70 level. (C) Immunostaining of Kv4.3 and Hsc70 in WT-NP and LP mice. Ventricular myocytes containing Kv4.3 (green) and Hsc70 (red); yellow indicates co-localization. The lower panels illustrate the OD along the white bar across the cells. Images are representative of at least 8–10 experiments. Scale bar, 20 µm. (D) Comparison of the plasma membrane to cytosol OD ratio between WT-NP and WT-LP mice. *p<0.001. WT-NP, wild-type non-pregnant; WT-LP, wild-type late-pregnant; OD, optical density.

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