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.
Highlights
Biomechanical remodeling is the process by which living tissues reorganize, reshape, and refit their microstructure in adaptation to changing internal and external forces
Atrioventricular (AV) valve morphogenesis is characterized by rapid extracellular matrix (ECM) accretion and turnover [5,6], which is hypothesized to be stimulated by a dynamic interaction of molecular and mechanical signaling
The ex vivo results were tested in ovo through an elevated 5-HT model. These results suggest that 5-HT may be an important potentiator of TGFb3 signaling in embryonic valve morphogenesis and biomechanical stiffening
Summary
Biomechanical remodeling is the process by which living tissues reorganize, reshape, and refit their microstructure in adaptation to changing internal and external forces. During post-EMT, these mesenchymal cells facilitate a transition in the cushion microstructure from glycosaminoglycans (GAGs) (hyaluronan, versican) toward fibrous structural proteins (collagen I, IV, V, fibronectin, periostin) [5,20,21] This shift in ECM content translates into increased valve stiffness [22], and coincides with elevated expression of TGFb3 in the cushions and AV canal [23]. A recent study reported TGFb1 upregulation in murine SERT KO hearts at near fetal stages, which was hypothesized to be a consequence of excess 5-HT signaling due to SERT inhibition [43] In light of these signaling interactions in both adult and development models, we hypothesize that this mechanically relevant crosstalk of TGFb and 5-HT may play a role in modulating embryonic AV cushion biomechanics. These results suggest that 5-HT may be an important potentiator of TGFb3 signaling in embryonic valve morphogenesis and biomechanical stiffening
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