Fig 2: Generation of PSA-deficient double KO mice by CRISPR/Cas9. (A) ST8Sia2 and ST8Sia4 sgRNA (blue) targeting sequences. Stop codon is labeled with a star and protospacer adjacent motif (PAM) sequence is indicated in red. Black arrowheads indicate the site of the mutation, which is indicated in parentheses for each. Base pair sequence indicates the region where the mutation is, which is indicated with an arrow in the KO sequences by Sanger sequencing. (B) Western blot of protein extract from whole ventricles from PSA-deficient DKOs and littermate controls at P1 (10 µg). Results show that PSA is not detected in DKO mice but is still detected in Sia2+/-/Sia4+/-, Sia2-/-/Sia4+/- and Sia2+/-/Sia4-/- littermates. Probing with anti-NCAM-1 antibody reveals unaffected NCAM-1 levels for all heart samples. Vinculin was used as loading control. (C) Body weight and heart weight/tibia length (HR/TL) ratios of Ctrl (DHet) and PSA-deficient DKO hearts. Hematoxylin and Eosin and trichrome stain of Ctrl (DHet) and PSA-deficient DKO hearts is shown below. Data represent mean±s.e.m. ***P<0.001, ****P<0.0001 (two-tailed Student's t-test) of Ctrl (DHet) versus DKO. Scale bar: 1 mm.

Fig 3: Ncam-1-deficient mice display ventricular conduction slowing and a dysmorphic VCS morphology. (A) Representative surface ECG traces of adult Cntn2, Alcam and Ncam-1 WT and KO mice. Ncam-1 KO mice show a modest but significant prolongation of the QRS interval compared with Ncam-1 WT controls. No difference in QRS was observed with Alcam- or Cntn2-deficient mice. (n=12 for each genotype). (B) Adult Cntn2, Alcam and Ncam-1 KO mice backcrossed into the Cntn2-EGFP reporter line were used to visualize the right and left HPS. Center panels are high-magnification views of the boxed areas of the left ventricle (LV). Right ventricle (RV) is displayed in the right-hand panels (Cntn2 and Alcam KO n=5; Ncam-1 KO n=9, of which six had overt morphological defects). (C) RFP+ cells dyed with TMRM were purified into ventricular (VM) and Purkinje (PC) fractions. Shown are representative FACS plots of PC (RFP+GFP+) and VM (RFP+GFP-) populations and the percentages of PCs relative to VMs were calculated for Ncam-1 WT and KO samples (n=4). (D) PC population was also quantified using MATLAB in acquired GFP images from WT and Ncam-1 KO hearts. Pixel density was calculated per unit of masked region. All comparisons between WT and KO groups were made by applying a two-tailed Student's t-test (n=4). Data represent mean±s.e.m. *P<0.05, ***P<0.001, WT versus KO. Scale bars: 1 mm.

Fig 4: Spatiotemporal expression of NCAM-1 and ALCAM during myocardial development. (A) Immunofluorescence staining of NCAM-1 (red) and Cx40 (green) during ventricular murine development at E10.5, E13.5 and E16.5. Right-hand panels show high-magnification views of the boxed areas. NCAM-1 is first detected in the compact myocardium at E13.5. By E16.5, NCAM-1 is expressed in the compact myocardium and a subset of Cx40+ cells in the trabecular myocardium. (B) Immunofluorescence staining for ALCAM (red) and Cx40 (green) during ventricular murine development at E10.5, E13.5 and E16.5. Right-hand panels show high-magnification views of the boxed areas. ALCAM is detected as early as E10.5 and its expression remains restricted to the trabecular myocardium. Scale bars: 100 µm (main panels); 25 µm (right-hand panels). (C) Immunofluorescence staining at P1 for NCAM-1 (green) and ALCAM (red) detected expression in trabecular ventricular myocardium, which becomes progressively restricted to the subendocardial region. By P7, NCAM-1 and ALCAM are restricted to PCs and absent from VMs. LV, left ventricle; RV, right ventricle. Scale bars: 25 µm.

Fig 5: PSA is expressed in the developing ventricular myocardium. (A) Western blot analysis of protein extract from whole ventricles at the specified developmental stages using anti-NCAM-1 and anti-PSA-NCAM-1 antibodies. Vinculin was used as loading control. Lysate protein amount loaded: Heart-PSA blot, 15 µg; Heart-NCAM-1 blot, 20 µg. (B) Immunofluorescence staining of NCAM-1 (red) and PSA (green) during ventricular murine development at E13.5, E14.5 and E16.5. PSA is first detected in the atria, AV junction and epicardium at E13.5. By E14.5 and for the remainder of embryonic development, PSA is also detected in NCAM-1+ trabecular cardiomyocytes in the subendocardial region. On the right, high-magnification images of the boxed area at E16.5 shows that PSA (green) and NCAM-1 (red) colocalize in the cell membrane. (C) Immunofluorescence staining of E16.5 Ncam-1 KO sequential sections shows no PSA expression in the absence of NCAM-1. (D) Tyrosine hydroxylase expression in AV junction at P3. Immunofluorescence staining of NCAM-1 (red) with TH (green) and PSA (green) at P3 showing atrioventricular junction region. Right-hand panels show high-magnification views of the boxed areas. AV, atrioventricular junction; LV, left ventricle; RV, right ventricle. Scale bars: 100 µm (B, main panels; C; D, left); 25 µm (B, high-magnification panel; D, right).