Serotonin 2B receptor is required for heart development

Abstract

Several lines of evidence suggest that the serotonin (5-hydroxytryptamine, 5-HT) regulates cardiovascular functions during embryogenesis and adulthood. 5-HT binds to numerous cognate receptors to initiate its biological effects. However, none of the 5-HT receptor disruptions in mice have yet resulted in embryonic defects. Here we show that 5-HT(2B) receptor is an important regulator of cardiac development. We found that inactivation of 5-HT(2B) gene leads to embryonic and neonatal death caused by heart defects. 5-HT(2B) mutant embryos exhibit a lack of trabeculae in the heart and a specific reduction in the expression levels of a tyrosine kinase receptor, ErbB-2, leading to midgestation lethality. These in vivo data suggest that the Gq-coupled receptor 5-HT(2B) uses the signaling pathway of tyrosine kinase receptor ErbB-2 for cardiac differentiation. All surviving newborn mice display a severe ventricular hypoplasia caused by impaired proliferative capacity of myocytes. In adult mutant mice, cardiac histopathological changes including myocyte disarray and ventricular dilation were consistently observed. Our results constitute genetic evidence that 5-HT via 5-HT(2B) receptor regulates differentiation and proliferation of developing and adult heart. This mutation provides a genetic model for cardiopathy and should facilitate studies of both the pathogenesis and therapy of cardiac disorders in humans.

Figure 1

Targeted disruption of the 5-HT_2B receptor gene. (a) (Top) The restriction map of the 5-HT_2B receptor genomic locus of interest; (Middle) the targeting construct; and (Bottom) the mutated locus after homologous recombination. (b) The wild-type (+/+), heterozygous (+/−), and homozygous mutant (−/−) alleles were detected by Southern blot analysis. The probe (P) located outside of the construct detects a 9.3-kbp BglII fragment from wild-type DNA whereas it is reduced to 3.2-kbp in the homologous recombined allele. (c) The analysis of receptor membrane proteins by binding assay revealed a complete loss of 5-HT_2B-specific sites in stomach and heart tissues of mutant mice. The maximal amount of binding sites is expressed as fmol specific binding/mg tissues ± SEM (n > 4). ND, no specific binding detected. (d) Genotyped progeny from 5HT_2B +/− intercrosses showed that the homozygous genotype did not exhibit a Mendelian ratio, indicating some embryonic lethality among the homozygous. The percent of progeny is calculated from 120 newborn mice from 14 different litters. The homozygous mutant (−/−) (22 out of 120) represented 66% of the expected viable calculated, assuming sum of +/+ and +/− is 75%. (e) Percentage of homozygous survival before and after the birth in the progeny of heterozygous crosses. Before birth (B), the percent of survival is estimated from more than four litters of heterozygous crosses (n > 30), and a Mendelian ratio is observed until 10.5 dpc.

Figure 2

Morphology of 5-HT_2B mutant embryos. (a) 5-HT_2B-deficient embryos at 10.5 dpc exhibit a typical bleeding into the pericardial cavity. (b) At 12.5 dpc, the mutant embryos are smaller and paler than their wild-type littermates. (c) Whole-mount immunohistochemical staining of 10.5 dpc embryos for developing blood vessels with platelet endothelial cell adhesion molecule antibody revealed no gross defects in vascular pattering compared with age-matched wild-type littermates but reduced staining in heart ventricle (v). (d) Semithin sagittal sections of 9.5 dpc embryos demonstrate a severe reduction in the thickness of the ventricle (v) including both the compact zone and the trabeculae in 5-HT_2B-deficient embryos. a, Atrium; en, endocardium. (e) Higher magnification shows a reduction of trabecular cells (tr) in the mutant heart, whereas elongated cells (white arrowhead) are visible in the compact subepicardial layer (sep). Epicardial cells (ep) are normally developed. (f) TEM analysis of these embryos reveals the abnormal sarcomeric differentiation within the subepicardial layer in all observed mutant heart (n = 6) but not in wild-type heart. z, Z band of the sarcomeres; f, actin fibers; m, mitochondria; and n, nucleus. Genotype designations are +/+, wild type; and −/−, homozygous mutant. (Bars for a–c = 500 μm; d = 100 μm; e = 5 μm, and f = 0.5 μm.)

Figure 3

Reduced ErbB-2 expression in 5-HT_2B mutant embryos. (a) The immunohistochemical staining of paraffin sections with anti-ErbB-2 antibody through the heart region of 10.5 dpc wild-type (Right) and mutant embryos (Left). (b) Enlargement of a. The ErbB-2 expression is globally reduced, including over the trabeculae (tr) and the compact zone (c) of the mutant ventricle (v). (c) Immunohistochemical labeling with an anti-5-HT_2B-specific antibody performed on cryostat sections of 10.5-dpc embryos illustrates the wild-type expression of 5-HT_2B receptor in the trabecular and the compact zone of the heart but not in the endocardial cushions (ec). (d) Semiquantitative reverse transcription–PCR shows a strong reduction of ErbB-2 mRNA in 9.5-dpc mutant embryos whereas ErbB-4 and the ribosomal elongation factor EF1a expression in the same RNA preparation remains unchanged. (e) A Western blot analysis with an anti-ErbB-2 antibody demonstrates a reduction of ErbB-2 expression in total protein extract from 5-HT_2B mutant embryos (from 9.5 to 11.5 dpc) (Upper). The platelet-derived growth factor receptor immunoreactivity in the same mutant embryo extracts remains unmodified (Lower). a, Atrium; α, anti-. (Bars for a and c = 250 μm, and b = 25 μm.)

Figure 4

Heart defects in postnatal 5-HT_2B mutant mice. (a) Histological analysis of newborn mutant hearts shows an obvious loss of ventricular (v) mass (hypoplasia). (b) Thymidine incorporation of isolated cardiomyocytes from newborn heart in response to 5-HT and neuregulin (heregulin) is significantly reduced in the 5-HT_2B mutant, indicating that myocyte proliferation is impaired and this effect is cell autonomous. The thymidine incorporation rate is expressed as fold increase over basal ± SEM (−/−, n = 3; and +/+, n = 3). (c) In vivo, BrdUrd incorporation (green) is almost undetectable in the heart of 5-HT_2B newborn mutant (Left), whereas BrdUrd incorporation was observed in the compact zone of control newborn heart (Right). Staining with 4′,6-diamidino-2-phenylindole of the same sections localizes the cell nuclei (blue). (d Upper) Semithin sections of heart in 6-week-old mutant shows abnormal morphology, including degenerating fiber (d) (Left). (d Lower) TEM of ultrathin sections shows that, in mutant heart, the sarcomere lengths are greatly reduced, the M-lines and the I-bands are indistinguishable, and the A-bands occupy the entire length between Z-bands (z) which themselves are thickened (Left) compared with wild-type age-mated heart (Right). m, Mitochondria. (Bars for a = 100 μm; c and d Upper = 20 μm; and d Lower = 0.5 μm.)