Influence of blood flow on cardiac development

Abstract

The role of hemodynamics in cardiovascular development is not well understood. Indeed, it would be remarkable if it were, given the dauntingly complex array of intricately synchronized genetic, molecular, mechanical, and environmental factors at play. However, with congenital heart defects affecting around 1 in 100 human births, and numerous studies pointing to hemodynamics as a factor in cardiovascular morphogenesis, this is not an area in which we can afford to remain in the dark. This review seeks to present the case for the importance of research into the biomechanics of the developing cardiovascular system. This is accomplished by i) illustrating the basics of some of the highly complex processes involved in heart development, and discussing the known influence of hemodynamics on those processes; ii) demonstrating how altered hemodynamic environments have the potential to bring about morphological anomalies, citing studies in multiple animal models with a variety of perturbation methods; iii) providing examples of widely used technological innovations which allow for accurate measurement of hemodynamic parameters in embryos; iv) detailing the results of studies in avian embryos which point to exciting correlations between various hemodynamic manipulations in early development and phenotypic defect incidence in mature hearts; and finally, v) stressing the relevance of uncovering specific biomechanical pathways involved in cardiovascular formation and remodeling under adverse conditions, to the potential treatment of human patients. The time is ripe to unravel the contributions of hemodynamics to cardiac development, and to recognize their frequently neglected role in the occurrence of heart malformation phenotypes.

Keywords: Congenital heart disease; Heart formation; Hemodynamics; Mechanotransduction.

Figure 1. Cardiac formation stages

(A) Cardiogenic cords, (B) linear tubular heart, (C) looped tubular heart, (D) cardiac septation, and (E) fully formed four-chambered heart. OFT: outflow tract, AVC: atrioventricular canal, C: endocardial cushion, V: primitive ventricle, T: trabeculae, A: primitive atrium, RA: right atrium, LA: left atrium, RV: right ventricle, LV: left ventricle, IVS: interventricular septum, PV: pulmonary valve, PA: pulmonary artery, MV: mitral valve, AoV: aortic valve, TV: tricuspid valve. The roman numbers in (C) and (D) correspond to the numbers assigned to the pharyngeal arch arteries (6 in total over developmental stages).

Figure 2. Schematics of endothelial-to-mesenchymal transition (EMT)

Left: schematic representation of the looped tubular heart depicting cushions in the outflow tract (OFT) and atrioventricular canal (AVC), as well as trabecular structures in the primitive ventricle (V). Inset: details of EMT in the AVC cushions, wherein a subset of activated endothelial cells (navy) delaminate from the endocardium, elongate, develop filopodia, migrate into the cardiac jelly, which is composed of extracellular matrix including some fibril proteins, and acquire a mesenchymal phenotype (pink). See Table 1 for relevant developmental periods.

Figure 3. Surgical Manipulations

Illustrations of (A) VVL in an HH17 avian embryo (B) LAL in an HH21 avian heart, (C) OTB in an HH18 avian heart, (D) microbead outflow occlusion and (E) microbead inflow occlusion in a 57 hpf zebrafish heart. See Table 1 for staging details. Illustrations adapted from Midgett 2014 and Hove et al 2003, with reference to videos from (Al Naieb et al., 2013). Blue shapes indicate sutures/clips/beads, red lines indicate blood flow. A lone bar-headed line indicates complete occlusion (A,D, and E) and a bar-headed line paired with a dashed arrow-headed line (B, C) indicates partial occlusion and perturbed flow. H: heart, E: eye, AVC: atrioventricular canal, OFT: outflow tract, V: ventricle, RA: right atrium, LA: left atrium, SV: sinus venosis, A: atrium.

Figure 4. Characterizing Hemodynamics

(A) Ultrasound trace for 1) untreated and 2) ethanol-injected HH19 avian embryos. Reproduced from (Peterson et al., 2017). (B1) video microscopy frame of a 3 dpf zebrafish heart, overlaid with 2D velocity vector field calculated using PIV; (B2) 2D WSS distribution calculated using the velocities from (B1). Reproduced from (Jamison et al., 2013). (C1) surface reconstructed from a confocal microscopy z-stack of an HH14 avian embryo; (C2) confocal microscopy image, overlaid with 3D velocity vector field calculated using PIV and validated with CFD performed using the mesh from (C1). Reproduced from (Hierck et al., 2008). (D1) OCT structural image of an HH18 avian outflow tract; (D2) concurrently collected Doppler OCT image (both overlaid with blue curves delineating the myocardium). Adapted from (Midgett et al., 2014). WSS: wall shear stress, OFT: outflow tract, V: ventricle, AVC: atrioventricular canal, A: atrium, dpf: days post fertilization.

Figure 5. Schematics of experimental design

Blood flow was perturbed at HH18 through VVL or OTB, and resulting blood flow dynamics were measured 2 hours after intervention. Embryos were then reincubated to HH24 (~24 hours after intervention) and either collected for further analysis or had their hemodynamics restored (for OTB embryos). Embryos that were not collected were then re-incubated and analyzed for structural cardiac malformations at HH38, when the heart was fully formed.

Figure 6. Measured hemodynamics after interventions

(A) Maximum blood velocity after interventions. Interventions performed at HH18; band removed at HH24. Constriction of banded embryos ranged between 21 and 52% tightness. Standard deviation is displayed as error bars. Asterisk: statistically significant differences between experimental and control embryos (n=8, p<0.05). (B) Altered hemodynamics after outflow tract banding. Hemodynamic response to outflow tract band tightness, produced from previously published data (Shi et al., 2013; Midgett et al., 2014). Vertical lines outline ranges of constriction used for further analysis, and standard deviation of controls are displayed as error bars. CON: surgical sham control, VVL: vitelline vein ligated, OTB: outflow tract banded. Reproduced from (Midgett et al., 2017b).

Figure 7. EMT vs band tightness in chicken embryos at HH24 with banding performed at HH18

(A) Cushion cell density and cell count per endocardium length quantitated from DAPI stain imaged with confocal microscopy. (B) Endocardial cell junctions per endocardium length quantitated from confocal immunofluorescent images stained for VE-cadherin. Reproduced from (Midgett et al., 2017a).

Figure 8. Cardiac defects depend on the level of hemodynamic perturbation

(A) Overall defect incidence among surviving embryos. Embryos were grouped by band tightness, VVL intervention, and controls were also included. (B) Separate defect type incidence among surviving embryos, by embryo group. CON: normal control, OTB: outflow tract banded embryos, VVL: vitelline vein ligated embryos, VSD: ventricular septal defect, CV VSD: conoventricular VSD, PM VSD: perimembranous VSD, M VSD: muscular VSD, DORV: double outlet right ventricle, TOF: Tetralogy of Fallot. Reproduced from (Midgett et al., 2017b).