Figure 4

(A) Confocal images of 48–60 hpf tie2:EGFP⁺ (labels valves green) hearts stained immunohistochemically for myosin (red) and either Ctsk or Ctss (blue) show cathepsin proteases are expressed in multiple regions of heart, with Ctsk enriched in cardiomyocytes (see insets of boxed regions) throughout ventricle and atria and Ctss predominantly present in region between the 2 chambers (see inset of boxed regions). n = 15–25 embryos imaged per condition. Scale bar: 20μm. V, ventricle; A, atrium. (B) qPCR of FAC sorted EGFP⁺ cardiomyocytes (cmlc:EGFP) and endocardial (tie2:EGFP) cells. Transcript abundance calculated relative to rpl4. Data are presented as mean ± SEM (C) Gel-based RT-PCR analyses of cathepsin expression in FACS-sorted cells. n = 50–100 embryos per sort. Representative gel from 3–4 independent experiments. (D) Confocal images of cryosections from 60–72 hpf tie2:EGFP⁺ (labels valves green) hearts stained immunohistochemically for myosin (red) and Ctsk (blue). n = 15 embryos imaged per condition. Scale bar: 20 μm. (E) Schematic illustrates cardiac injection of an activity-based probe (ABP), which does not fluoresce until covalently bound to an activated cysteine cathepsin. Confocal images of WT and gnptab morphants (MO) labeled with the pan reactive probe BMV109 reveal generally increased cathepsin activity (red, denoted by white arrowheads) present throughout the MLII heart. Injection of the Ctss-specific probe BMV157 (red) shows its activity is reduced in the MLII heart compared with other cathepsins, likely K and L. Representative images from 3 independent experiments, n = 30 embryos per condition. Scale bar: 30 μm. OFT, outflow tract.