Application of vasopressin causes vasoconstriction of major blood vessels in zebrafish by 48 hpf. (A) RT-PCR of vasopressin receptors in whole body vs. cardiac tissues. (B) Diagram of zebrafish vasculature. The orange line indicates site of measurement. (C) Vasculature of control and (C’) vasopressin-treated embryos at 48 hpf. (D) Diameter of cardinal vein (CV), dorsal aorta (DA), and intersegmental vessels (ISV). For D: n = 11 for wildtype, n = 11 for vasopressin-treated embryos. Two-sided t-test was performed to validate statistical significance; * indicates p < 0.05. Scale bar represents 50 µm.

Increased pressure causes chamber remodeling by 56 hpf. (A) Cardiac output (CO) at 40 hpf at increasing doses of vasopressin (control, 2.5, 5, 7.5, and 10 µm). (B) CO at 56 hpf at increasing doses of vasopressin (vaso). n = 5 fish per group. (C) Cardiac output at 40 and 56 hpf in control and 10 µm vaso-treated fish. Each colored dot represents an individual fish; orange-colored dots represent control fish, light blue represents mildly-affected fish, and purple represents severely-affected fish. n= 4 control, 8 vaso. (D) Rate of change for cardiac output with respect to time. (E) Stroke volume and (F) heart rate of control and 10 µm vaso-treated fish at 40 and 56 hpf. n = 11 control and 12 vaso-treated fish at 40 hpf, and 8 and 13 vasopressin-treated fish at 56 hpf. * indicates p < 0.05

Increased pressure causes hypertrophy of ventricular outer curvature (OC) myocytes by 56 hpf. (A) Control and (B) vaso-treated hearts showing z-disks (A,B) and cell shape through ALCAM immunohistochemistry (A′,B′). (A′′,B′′) display merged images. Purple box shows regions of interest shown in (A′′′,B′′′). For A–A′′, and B–B′′, scale bar represents 20 µm. (A′′′) Control and (B′′′) vaso-treated hearts. Scale bar represents 10 µm. (C) Area of ventricular OC cells in control and vaso-treated hearts. (D) Total z-disk size (TZB) of sarcomeres present in each cell normalized to the area of each cell (1/µm). For details for how TML was measured, see supplemental Figure S11. (E) Number of z-disks per cell in control and vaso-treated hearts. (F) Average length of the sarcomeres present in each cell. For control and vaso-treated hearts, 14 fish were analyzed. Five cells from the OC were analyzed per fish, such that 70 total cells were analyzed. Each colored dot on the graph above represents an individual fish. * indicates p < 0.05, Student’s t-test was used for statistical comparisons

Increased afterload increases retrograde flow and alters AVJ mechanics. (A) Diameter of the flow field at the atrioventricular junction in control hearts at 56 hpf. X–axis is normalized time across a cardiac cycle, where 0 corresponds to the beginning of atrial contraction and 1 represents the end of ventricular contraction. n = 4 (B) Diameter analysis of vaso-treated hearts that showed hypertrophic growth between 40 and 56 hpf. n = 6 (C) Diameter analysis of vaso-treated hearts at 56 hpf. These hearts showed severely altered pumping mechanics at 40 hpf. (D) Maximum diameter analysis of control, vaso-treated hearts that showed altered pumping (AP) and vaso-treated hearts that showed hypertrophic growth (HH). (E) Retrograde flow fraction (RFF) at 40 and 56 hpf in control and vaso-treated hearts. n = 11 control and 12 vaso-treated fish. * indicates p < 0.05.

Increased pressure alters AVJ myocyte morphology and specification. (A) Control and (B) vaso-treated hearts showing z-disks and cell shape through ALCAM immunohistochemistry at 56 hpf. Purple box shows region of interest shown in (A′,B′). For (A,B), scale bar represents 20 µm. (A′) Control and (B′) vaso-treated hearts. Scale bar represents 10 µm. White arrow heads indicate AVJ. (C) TZB of sarcomeres present in each cell normalized to the area of each cell (1/µm). n = 36 cells for control and 33 for vaso-treated hearts. (D) In situ hybridization (ISH) of myocardial AVJ marker bmp4 in control and (E,F) vaso-treated hearts. (D′–F′) Diagrams of ISH expression patterns. (G) Percent of fish expressing a particular bmp4 expression pattern. A total of 20 fish were scored in each group. * indicates p < 0.05.

Increased myocardial afterload causes aberrant AVJ cell specification. (A) Control hearts stained with ALCAM at 56 hpf. (A’) Control hearts stained with ALCAM at 72 hpf. Scale bar represents 20µm. (B,B’) Representative image of vaso-treated heart at (B) 56 hpf and at (B’) 72 hpf. (C) Number of ALCAM expressing endocardial cells. N = 11 wildtype hearts, 10 vaso treated hearts at 56 hpf. Statistical significance was determined using Student’s t-test; * indicates p < 0.05. (C,D) Number of ALCAM expressing endocardial cells. N = 11 wildtype hearts, 10 vaso-treated hearts at 72 hpf. Five cells were analyzed per location per heart, area was measured for each cell and averaged. AVJ myocardial cells were defined as myocardial cells located in the constriction between the atrium and ventricle.