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, 100 (23), 13525-30

Disruption of the Nonneuronal tph1 Gene Demonstrates the Importance of Peripheral Serotonin in Cardiac Function

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Disruption of the Nonneuronal tph1 Gene Demonstrates the Importance of Peripheral Serotonin in Cardiac Function

Francine Côté et al. Proc Natl Acad Sci U S A.

Abstract

Serotonin (5-HT) controls a wide range of biological functions. In the brain, its implication as a neurotransmitter and in the control of behavioral traits has been largely documented. At the periphery, its modulatory role in physiological processes, such as the cardiovascular function, is still poorly understood. The rate-limiting enzyme of 5-HT synthesis, tryptophan hydroxylase (TPH), is encoded by two genes, the well characterized tph1 gene and a recently identified tph2 gene. In this article, based on the study of a mutant mouse in which the tph1 gene has been inactivated by replacement with the beta-galactosidase gene, we establish that the neuronal tph2 is expressed in neurons of the raphe nuclei and of the myenteric plexus, whereas the nonneuronal tph1, as detected by beta-galactosidase expression, is in the pineal gland and the enterochromaffin cells. Anatomic examination of the mutant mice revealed larger heart sizes than in wild-type mice. Histological investigation indicates that the primary structure of the heart muscle is not affected. Hemodynamic analyses demonstrate abnormal cardiac activity, which ultimately leads to heart failure of the mutant animals. This report links loss of tph1 gene expression, and thus of peripheral 5-HT, to a cardiac dysfunction phenotype. The tph1-/- mutant may be valuable for investigating cardiovascular dysfunction observed in heart failure in humans.

Figures

Fig. 1.
Fig. 1.
Targeted disruption of the tph1 gene. (A) Strategy for targeting the tph gene. (Top) The endogenous tph1 locus. (Middle) The targeting construct. (Bottom) The structure after homologous recombination. (B) Southern blot analysis of the wild-type (+/+), heterozygous (+/-), and homozygous (-/-) alleles. The 5′ probe, as indicated in A, recognizes a sequence outside the recombined region. An 8-kb EcoRI fragment is detected in the wild-type allele, and a 5.4-kb fragment is detected in the recombined allele. (C) Immunohistochemical analysis of the pineal gland and the dorsal raphe nucleus of homozygous tph1 mutants. (Upper) β-Gal immunoreactivity. (Lower) TPH immunoreactivity as described (39). (Scale bars = 0.1 mm.)
Fig. 2.
Fig. 2.
Tissue-specific expression of tph1 and tph2.(A) 5-HT levels in tph1-/- and tph1+/+ mice in the brainstem (tph1-/-, n = 11, and tph1+/+, n = 8), the pineal gland (tph1-/-, n = 17, and tph1+/+, n = 8), the gut (tph1-/-, n = 10, and tph1+/+, n = 4), and the blood (tph1-/-, n = 7, and tph1+/+, n = 7). Values are expressed as mean ± SEM; *, P < 0.05; **, P < 0.001 (Student t test) for the difference between tph1-/- and tph1+/+ mice. (B) TPH1 and TPH2 mRNAs as revealed by ISH. The TPH1 riboprobe was used in Ba, Bb, and Be; the TPH2 riboprobe was used in Bc and Bd. The β-GAL riboprobe was used in Bf. (Scale bars = 1 mm.)
Fig. 3.
Fig. 3.
Cardiac defect phenotype of homozygous tph1 mutants. (A) Gross morphology of 26-week-old tph1-/- and tph1+/+ mice. (Scale bars = 1 mm.) (B-D) Cardiac function of 20-week-old mice as assessed by in vivo cardiac catheterization. (B) The mean values of cardiac output (microliters per minute) (formula image), stroke volume (microliters) (▪), and heart rates derived from the pressure input [beats per minute (bpm)] (□). Means for heart rate are: tph1+/+, 266 ± 23 bpm; tph1-/-, 336 ± 53 bpm; n = 6 and P = 0.007. (C) Distribution of individual dP/dtmax values measuring the maximum rate of the increase of the LV pressure. (D) Mean diastolic (▪) and systolic (formula image) blood pressure as expressed in mmHg in the steady state (n = 6).
Fig. 4.
Fig. 4.
Analysis of pressure-volume loops series and the histology of two tph1-/- mutants representing extreme cases with a wild-type animal for reference (#X2). Mouse #273 compensates, and mouse #296 decompensates. (Scale bars = 1 mm.)

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