Inappropriate cathepsin K secretion promotes its enzymatic activation driving heart and valve malformation
Po-Nien Lu, Trevor Moreland, Courtney J. Christian, Troy C. Lund, Richard A. Steet, Heather Flanagan-Steet
Po-Nien Lu, Trevor Moreland, Courtney J. Christian, Troy C. Lund, Richard A. Steet, Heather Flanagan-Steet
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Research Article Cell biology Development

Inappropriate cathepsin K secretion promotes its enzymatic activation driving heart and valve malformation

Abstract

Although congenital heart defects (CHDs) represent the most common birth defect, a comprehensive understanding of disease etiology remains unknown. This is further complicated since CHDs can occur in isolation or as a feature of another disorder. Analyzing disorders with associated CHDs provides a powerful platform to identify primary pathogenic mechanisms driving disease. Aberrant localization and expression of cathepsin proteases can perpetuate later-stage heart diseases, but their contribution toward CHDs is unclear. To investigate the contribution of cathepsins during cardiovascular development and congenital disease, we analyzed the pathogenesis of cardiac defects in zebrafish models of the lysosomal storage disorder mucolipidosis II (MLII). MLII is caused by mutations in the GlcNAc-1-phosphotransferase enzyme (Gnptab) that disrupt carbohydrate-dependent sorting of lysosomal enzymes. Without Gnptab, lysosomal hydrolases, including cathepsin proteases, are inappropriately secreted. Analyses of heart development in gnptab-deficient zebrafish show cathepsin K secretion increases its activity, disrupts TGF-β–related signaling, and alters myocardial and valvular formation. Importantly, cathepsin K inhibition restored normal heart and valve development in MLII embryos. Collectively, these data identify mislocalized cathepsin K as an initiator of cardiac disease in this lysosomal disorder and establish cathepsin inhibition as a viable therapeutic strategy.

Authors

Po-Nien Lu, Trevor Moreland, Courtney J. Christian, Troy C. Lund, Richard A. Steet, Heather Flanagan-Steet

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Figure 4

Cathepsin proteases are expressed in developing hearts.

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Cathepsin proteases are expressed in developing hearts. (A) Confocal ima...
(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.