Fluid forces shape the embryonic heart: Insights from zebrafish

Abstract

Heart formation involves a complex series of tissue rearrangements, during which regions of the developing organ expand, bend, converge, and protrude in order to create the specific shapes of important cardiac components. Much of this morphogenesis takes place while cardiac function is underway, with blood flowing through the rapidly contracting chambers. Fluid forces are therefore likely to influence the regulation of cardiac morphogenesis, but it is not yet clear how these biomechanical cues direct specific cellular behaviors. In recent years, the optical accessibility and genetic amenability of zebrafish embryos have facilitated unique opportunities to integrate the analysis of flow parameters with the molecular and cellular dynamics underlying cardiogenesis. Consequently, we are making progress toward a comprehensive view of the biomechanical regulation of cardiac chamber emergence, atrioventricular canal differentiation, and ventricular trabeculation. In this review, we highlight a series of studies in zebrafish that have provided new insight into how cardiac function can shape cardiac morphology, with a particular focus on how hemodynamics can impact cardiac cell behavior. Over the long-term, this knowledge will undoubtedly guide our consideration of the potential causes of congenital heart disease.

Keywords: Atrioventricular canal; Blood flow; Cardiac chambers; Heart development; Trabeculation.

Figure 1:. Analysis of blood flow in the zebrafish.

(A) Labeled erythrocytes in the dorsal aorta of an embryo carrying the transgene Tg(gata1:dsRed) are visualized at a single timepoint. (B) Image segmentation identifies individual erythrocytes (green dots). (C) A tracking algorithm reveals the trajectories (yellow lines) of the erythrocytes, allowing measurement of their displacement and estimation of their velocity. (D) Workflow options for analysis of blood flow in zebrafish. PIV, particle image velocimetry; CFD, computational fluid dynamics; WSS, wall shear stress; RFF, retrograde flow fraction. Panels A-C adapted from Watkins et al., 2012.

Figure 2:. Cardiac function modulates cardiomyocyte dimensions in the ventricle.

Confocal projections show the ventricle in embryos carrying the transgene Tg(myl7:egfp), with mosaic expression of Tg(myl7:dsRedt4). (A,C) At 52 hours post-fertilization (hpf), cardiomyocytes in the outer curvature of the ventricle (arrows) have acquired a characteristic size and an elongated morphology. (B) In weak atrium (wea) mutants, blood flow into the ventricle is reduced, and the cardiomyocytes in the outer curvature are abnormally small (arrow) and circular (arrowhead). (D) In half-hearted (haf) mutants, the ventricle is non-contractile, and the outer curvature cardiomyocytes are abnormally large and distended (arrows). Images adapted from Auman et al., 2007.

Figure 3:. Fluid forces influence endocardial cell number during chamber emergence.

(A-D) Endocardial cells (green) are indicated by expression of the transgene Tg(kdrl:GFP); localization of Amhc (blue) and actin (red) are also shown. Whereas wild-type embryos have ~80 and ~70 endocardial cells in the ventricle and the atrium, respectively (A,E), the number of endocardial cells in both chambers is significantly reduced in silent heart (sih; tnnt2a) mutants (B,E), in which the heart is noncontractile. gata1/2 morphants, which have diminished shear forces due to their reduced hematocrit, display fewer endocardial cells in the atrium (C,E). In contrast, gata2 morphants, which have been shown to have reduced retrograde flow in addition to diminished shear forces, display fewer endocardial cells in both chambers (D,E), similar to sih mutants. (F) Consistent with this, endocardial proliferation in the ventricle is significantly reduced in sih mutants and gata2 morphants, but not in gata1/2 morphants. Images adapted from Dietrich et al., 2014.

Figure 4:. Cardiac function regulates endocardial convergence at the AVC.

(A) The AVC endocardium can be subdivided into four regions: superior (a), exterior (b), inferior (c) and interior (d). (B-E) Representations of the unfolded endocardium in Tg(fli:kaede) embryos in which the AVC endocardium has been labeled via photoconversion. In wild-type embryos, the AVC endocardial cells converge between 36 hpf (B) and 48 hpf (C). In contrast, in sih mutants, the AVC endocardium widens between 36 hpf (D) and 48 hpf (E). Images adapted from Boselli et al., 2017.

Figure 5:. Interplay between fluid forces and ventricular trabeculation.

Representations of the inner surface of the ventricle, overlaid with a depiction of the oscillatory shear index (OSI) at 4 dpf. (A) In wild-type, the OSI is relatively high in trabecular grooves and relatively low in trabecular ridges. (B) In AG1478-treated embryos, the Neuregulin signaling pathway is inhibited, resulting in reduced trabeculation and a smoother inner surface of the ventricle. A similar reduction of trabeculation is observed in gata1a morphants (C) and wea mutants (D). Images adapted from Vedula et al., 2017.

Figure 6:. Mechanoresponsive pathways regulating cardiac development in zebrafish.

During AVC development, oscillatory flow activates mechanosensitive calcium channels Trpp2 and Trpv4 in the endocardium, which then promote klf2a expression (Heckel et al., 2015). In addition, flow-induced Heg1 represses klf2a expression in conjunction with the CCM complex, establishing a feedback loop for klf2a (Donat et al., 2018; Renz et al., 2015). Klf2a, in turn, regulates wnt9b expression in the endocardium (Goddard et al., 2017) and Fibronectin deposition in the extracellular matrix (ECM) (Steed et al., 2016). At the same time, flow-induced miR-21 represses receptor tyrosine kinase signaling by targeting sprty2 (Banjo et al., 2013). Meanwhile, during ventricle emergence, function-dependent miR-143 inhibits retinoic acid signaling in the endocardium (Miyasaka et al., 2011) and targets Adducin3 in the myocardium (Deacon et al., 2010). Mechanical forces also activate Notch signaling in the endocardium, which further induces expression of the Erbb2 ligand nrg1 (Samsa et al., 2015). During trabeculation, cardiac function regulates nrg2a (Rasouli and Stainier, 2017) and erbb2 expression (Lee et al., 2016), as well as Wwtr1 localization (Lai et al., 2018). The Nrg/Erbb2 pathway can further affect Notch signaling in cardiomyocytes (Jiménez-Amilburu et al., 2016) and regulate Wwtr1 localization, which can also activate Notch signaling (Lai et al., 2018).