Effect of left atrial ligation-driven altered inflow hemodynamics on embryonic heart development: clues for prenatal progression of hypoplastic left heart syndrome

Fig. 1. In-ovo chick embryo culture enables access to surgeries and analysis. a Embryos are cultures within their shells and opened at embryonic day (ED) 3. The egg opening creates an easy access for b surgery and c echocardiographic imaging, respectively

Fig. 2. LAL surgery. a Schematic representation of LAL steps. b Representative pictures of LAL steps. Step 1 Opening chorionic and allantoic membranes and exposing the animal. Step 2 Flipping the animal vertically to expose its left side shown in Step 3. Step 4 Opening the pericardium and exposing the left atrium. Step 5 Placing the pre-prepared surgical knot on the top of the atrium. Step 6 Tightening the knot around the atrium. Step 7 Cutting the extra ends of the suture and finally flipping back the animal to its original orientation

Fig. 3. Echocardiography imaging for an HH21 embryo. B-mode images and Doppler velocity measurements for AV canal. In B-mode images, AVC is atrioventricular cushion. Arrow shows the blood flow direction. Edges of atrial and ventricular myocardium are highlighted in black. Edges of AVC are highlighted in yellow. Doppler blood velocity profile shows two peaks which correspond to initial ventricular relaxation and atrial contraction/ventricular filling phases

Fig. 4. Micro-CT cast creation and 3D volume generation. a CT-dense contrast agent is perfused through cardiovascular system for cast creation. Micro-CT scanning enables generation of 3D heart volumes. b Unseptated 3.5-day (HH21) heart. c Septated 7-day (HH30) heart. d Orifice sizes for right and left AV canals in c can be seen through a section inside atria. RAV right atrioventricular canal, LAV left atrioventricular canal, R right, L left, RA right atria, LA left atria, RV right ventricle, LV left ventricle

Fig. 5. 3D geometries and sectioned models. a HH21 LAL hearts. b HH21 control hearts. c HH30 LAL hearts. d HH30 control hearts. Left atria is outlined by dash line for all embryonic hearts. LA, LV, RA, RV are sectioned to apply the inlet and outlet boundary conditions. LA left atrium, RA right atrium, LV left ventricle, RV right ventricle

Fig. 6. Hemodynamic profiles of LAL and control chicks. a HH21 AV canal velocity profiles. b HH30 L-AV canal velocity profiles. c HH30 R-AV canal velocity profiles. Results are presented as mean ± standard error of the mean (SEM). L-AV left atrioventricular canal, R-AV right atrioventricular canal

Fig. 7. Example of histologically H&E stained slides of embryonic chick hearts at different stages. a The orientation and anatomy of HH25 for control and LAL hearts. b The anatomy of HH30 for control and LAL hearts

Fig. 8. LAL causes anatomical changes on embryonic day-5 (ED5) chick hearts 24-h post-surgery. ED5 control and LAL hearts are oriented and H&E processed. a Quantification of length parameters (HH, HW, and MT) in both control and LAL hearts. b The quantification of area parameters (LV, SAVC, and IAVC) in both control and LAL hearts. HH heart height, HW heart width, MT myocardium thickness, LV left ventricle, SAVC superior atrioventricular cushion, IAVC inferior atrioventricular cushion. Data is presented as mean (n = 3), error bars indicate SEM, and p < 0.05 indicates statistical significance as determined by Student’s t test (*p < 0.05)

Fig. 9. LAL causes anatomical changes on ED7 chick hearts 72-h post-surgery. ED7 control and LAL hearts are oriented and H&E processed. Different parameters are measured using ImageJ. a Demonstration of the quantified parameters shown in b–g. h Demonstration of the parameters quantified in i–l. LA left atrium, LV left ventricle, RA right atrium, RV right ventricle. Data is presented as mean (n = 3), error bars indicate SEM, and p < 0.05 indicates statistical significance as determined by Student’s t test (*p < 0.05)

Fig. 10. Micro-CT geometries for control and LAL embryos at a HH21 and at b HH30. Left atrium regions are outlined for HH21 embryos. Left ventricle regions are shown by arrows for HH30 embryos

Fig. 11. WSS distribution at peak AV flow velocity for HH21 control hearts. Solid and dashed arrows show left and right AV canal regions, respectively. Red arrows designate inferior and superior AV cushions

Fig. 12. WSS distribution at peak AV flow velocity for HH21 LAL hearts. Solid and dashed arrows show left and right AV canal regions, respectively. Red arrows designate inferior and superior AV cushions