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Serotonin potentiates transforming growth factor-beta3 induced biomechanical remodeling in avian embryonic atrioventricular valves.

Buskohl PR, Sun MJ, Sun ML, Thompson RP, Butcher JT - PLoS ONE (2012)

Bottom Line: Blockade of TGFβ type I receptors via SB431542 inhibited the TGFβ3 effects.Elevated 5-HT in ovo resulted in elevated remodeling gene expression and increased TGFβ signaling activity, supporting our ex-vivo findings.Collectively, these results highlight TGFβ/5-HT signaling as a potent mechanism for control of biomechanical remodeling of AV cushions during development.

View Article: PubMed Central - PubMed

Affiliation: Department of Mechanical and Aerospace Engineering, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Embryonic heart valve primordia (cushions) maintain unidirectional blood flow during development despite an increasingly demanding mechanical environment. Recent studies demonstrate that atrioventricular (AV) cushions stiffen over gestation, but the molecular mechanisms of this process are unknown. Transforming growth factor-beta (TGFβ) and serotonin (5-HT) signaling modulate tissue biomechanics of postnatal valves, but less is known of their role in the biomechanical remodeling of embryonic valves. In this study, we demonstrate that exogenous TGFβ3 increases AV cushion biomechanical stiffness and residual stress, but paradoxically reduces matrix compaction. We then show that TGFβ3 induces contractile gene expression (RhoA, aSMA) and extracellular matrix expression (col1α2) in cushion mesenchyme, while simultaneously stimulating a two-fold increase in proliferation. Local compaction increased due to an elevated contractile phenotype, but global compaction appeared reduced due to proliferation and ECM synthesis. Blockade of TGFβ type I receptors via SB431542 inhibited the TGFβ3 effects. We next showed that exogenous 5-HT does not influence cushion stiffness by itself, but synergistically increases cushion stiffness with TGFβ3 co-treatment. 5-HT increased TGFβ3 gene expression and also potentiated TGFβ3 induced gene expression in a dose-dependent manner. Blockade of the 5HT2b receptor, but not 5-HT2a receptor or serotonin transporter (SERT), resulted in complete cessation of TGFβ3 induced mechanical strengthening. Finally, systemic 5-HT administration in ovo induced cushion remodeling related defects, including thinned/atretic AV valves, ventricular septal defects, and outflow rotation defects. Elevated 5-HT in ovo resulted in elevated remodeling gene expression and increased TGFβ signaling activity, supporting our ex-vivo findings. Collectively, these results highlight TGFβ/5-HT signaling as a potent mechanism for control of biomechanical remodeling of AV cushions during development.

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5-HT administration in ovo induces cardiac defects.A) Representative virtual sections of control, 5-HT treated, and thoracotomy sham control hearts at HH36 via endopainting and confocal microscopy. B) Representative Movat's pentachrome stained sections of hearts with the same conditions. Prominent cardiac defects, including enlarged atria (EA) and ventricular septal defect (VSD), were associated with malformed and malfunctioning AV valves (arrows). 25×, scale bar = 500 µm. C) Left septal leaflet average thickness and minimum thickness are both statistically thinner in 5-HT treated leaflets than control. mean ± SEM, n = 3–6 hearts per treatment, *p<0.05, t-test.
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pone-0042527-g006: 5-HT administration in ovo induces cardiac defects.A) Representative virtual sections of control, 5-HT treated, and thoracotomy sham control hearts at HH36 via endopainting and confocal microscopy. B) Representative Movat's pentachrome stained sections of hearts with the same conditions. Prominent cardiac defects, including enlarged atria (EA) and ventricular septal defect (VSD), were associated with malformed and malfunctioning AV valves (arrows). 25×, scale bar = 500 µm. C) Left septal leaflet average thickness and minimum thickness are both statistically thinner in 5-HT treated leaflets than control. mean ± SEM, n = 3–6 hearts per treatment, *p<0.05, t-test.

Mentions: As the effects of TGFβ signaling on valve formation are well studied [17], [18], [57], we here test whether exogenous 5-HT administration in ovo alters valve morphogenesis. 5-HT administration in ovo at HH17 induced a spectrum of cardiac defects by HH36 (Day 10) as summarized in Table 1. Temporal and dosage dependant viability curves (Figure S6A) showed that a 0.7 mg dose was over 50% lethal at HH36, but administration of the same dose of 5-HT at HH25 or HH31 did not result in further lethality or defect formation (data not shown). The only gross malformations observed were localized to the heart and chest wall. Approximately 42% (24/57) of affected embryos exhibited an ectopic heart which protruded through an incomplete chest wall closure (Figure S6B). To confirm that interior defects resulted specifically from 5-HT exposure and not secondarily from the ectopia, an experimental thoracotomy was performed to model the ectopic condition. We found no statistically significant occurrence of any cardiac defects with experimental ectopia, supporting that 5-HT was responsible for the cardiac defects observed. A ventricular septal defect (VSD or SVSD) occurred in 42% (24/57) of the defective embryos. Approximately 18% (10/57) of the embryos exhibited double outlet right ventricle (DORV) defects. 5-HT administration also resulted in significantly enlarged atria with thinned walls in 35% (20/57) of the defective embryos (Table 1, Figure 6A). All of the embryos with DORV also exhibited highly stenotic or atretic atrioventricular (AV) valves (Figure 6B), with the normally muscular flap valve in the right AV canal appearing thin and fibrous like the left AV valve. Regardless of gross cardiac defect identified, the average (0.144±0.009 mm, mean ± SEM) and minimal (0.080±0.007 mm) thickness of the left AV septal leaflet was thinner in 5-HT treated embryos than controls (0.191±0.009 and 0.165±0.023 mm respectively, Figure 6C). No differences were found in mural leaflet thickness, or in the length of either leaflet. The reduction in AV valve thickness with 5-HT treatment indicated an increase in tissue compaction, and may possibly be a recapitulation of the migratory/contractile phenotype observed ex vivo.


Serotonin potentiates transforming growth factor-beta3 induced biomechanical remodeling in avian embryonic atrioventricular valves.

Buskohl PR, Sun MJ, Sun ML, Thompson RP, Butcher JT - PLoS ONE (2012)

5-HT administration in ovo induces cardiac defects.A) Representative virtual sections of control, 5-HT treated, and thoracotomy sham control hearts at HH36 via endopainting and confocal microscopy. B) Representative Movat's pentachrome stained sections of hearts with the same conditions. Prominent cardiac defects, including enlarged atria (EA) and ventricular septal defect (VSD), were associated with malformed and malfunctioning AV valves (arrows). 25×, scale bar = 500 µm. C) Left septal leaflet average thickness and minimum thickness are both statistically thinner in 5-HT treated leaflets than control. mean ± SEM, n = 3–6 hearts per treatment, *p<0.05, t-test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412853&req=5

pone-0042527-g006: 5-HT administration in ovo induces cardiac defects.A) Representative virtual sections of control, 5-HT treated, and thoracotomy sham control hearts at HH36 via endopainting and confocal microscopy. B) Representative Movat's pentachrome stained sections of hearts with the same conditions. Prominent cardiac defects, including enlarged atria (EA) and ventricular septal defect (VSD), were associated with malformed and malfunctioning AV valves (arrows). 25×, scale bar = 500 µm. C) Left septal leaflet average thickness and minimum thickness are both statistically thinner in 5-HT treated leaflets than control. mean ± SEM, n = 3–6 hearts per treatment, *p<0.05, t-test.
Mentions: As the effects of TGFβ signaling on valve formation are well studied [17], [18], [57], we here test whether exogenous 5-HT administration in ovo alters valve morphogenesis. 5-HT administration in ovo at HH17 induced a spectrum of cardiac defects by HH36 (Day 10) as summarized in Table 1. Temporal and dosage dependant viability curves (Figure S6A) showed that a 0.7 mg dose was over 50% lethal at HH36, but administration of the same dose of 5-HT at HH25 or HH31 did not result in further lethality or defect formation (data not shown). The only gross malformations observed were localized to the heart and chest wall. Approximately 42% (24/57) of affected embryos exhibited an ectopic heart which protruded through an incomplete chest wall closure (Figure S6B). To confirm that interior defects resulted specifically from 5-HT exposure and not secondarily from the ectopia, an experimental thoracotomy was performed to model the ectopic condition. We found no statistically significant occurrence of any cardiac defects with experimental ectopia, supporting that 5-HT was responsible for the cardiac defects observed. A ventricular septal defect (VSD or SVSD) occurred in 42% (24/57) of the defective embryos. Approximately 18% (10/57) of the embryos exhibited double outlet right ventricle (DORV) defects. 5-HT administration also resulted in significantly enlarged atria with thinned walls in 35% (20/57) of the defective embryos (Table 1, Figure 6A). All of the embryos with DORV also exhibited highly stenotic or atretic atrioventricular (AV) valves (Figure 6B), with the normally muscular flap valve in the right AV canal appearing thin and fibrous like the left AV valve. Regardless of gross cardiac defect identified, the average (0.144±0.009 mm, mean ± SEM) and minimal (0.080±0.007 mm) thickness of the left AV septal leaflet was thinner in 5-HT treated embryos than controls (0.191±0.009 and 0.165±0.023 mm respectively, Figure 6C). No differences were found in mural leaflet thickness, or in the length of either leaflet. The reduction in AV valve thickness with 5-HT treatment indicated an increase in tissue compaction, and may possibly be a recapitulation of the migratory/contractile phenotype observed ex vivo.

Bottom Line: Blockade of TGFβ type I receptors via SB431542 inhibited the TGFβ3 effects.Elevated 5-HT in ovo resulted in elevated remodeling gene expression and increased TGFβ signaling activity, supporting our ex-vivo findings.Collectively, these results highlight TGFβ/5-HT signaling as a potent mechanism for control of biomechanical remodeling of AV cushions during development.

View Article: PubMed Central - PubMed

Affiliation: Department of Mechanical and Aerospace Engineering, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Embryonic heart valve primordia (cushions) maintain unidirectional blood flow during development despite an increasingly demanding mechanical environment. Recent studies demonstrate that atrioventricular (AV) cushions stiffen over gestation, but the molecular mechanisms of this process are unknown. Transforming growth factor-beta (TGFβ) and serotonin (5-HT) signaling modulate tissue biomechanics of postnatal valves, but less is known of their role in the biomechanical remodeling of embryonic valves. In this study, we demonstrate that exogenous TGFβ3 increases AV cushion biomechanical stiffness and residual stress, but paradoxically reduces matrix compaction. We then show that TGFβ3 induces contractile gene expression (RhoA, aSMA) and extracellular matrix expression (col1α2) in cushion mesenchyme, while simultaneously stimulating a two-fold increase in proliferation. Local compaction increased due to an elevated contractile phenotype, but global compaction appeared reduced due to proliferation and ECM synthesis. Blockade of TGFβ type I receptors via SB431542 inhibited the TGFβ3 effects. We next showed that exogenous 5-HT does not influence cushion stiffness by itself, but synergistically increases cushion stiffness with TGFβ3 co-treatment. 5-HT increased TGFβ3 gene expression and also potentiated TGFβ3 induced gene expression in a dose-dependent manner. Blockade of the 5HT2b receptor, but not 5-HT2a receptor or serotonin transporter (SERT), resulted in complete cessation of TGFβ3 induced mechanical strengthening. Finally, systemic 5-HT administration in ovo induced cushion remodeling related defects, including thinned/atretic AV valves, ventricular septal defects, and outflow rotation defects. Elevated 5-HT in ovo resulted in elevated remodeling gene expression and increased TGFβ signaling activity, supporting our ex-vivo findings. Collectively, these results highlight TGFβ/5-HT signaling as a potent mechanism for control of biomechanical remodeling of AV cushions during development.

Show MeSH
Related in: MedlinePlus