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 5

Treatment with the cathepsin K inhibitor odanacatib rescues MLII heart defects.

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Treatment with the cathepsin K inhibitor odanacatib rescues MLII heart d...
(A and B) Confocal analyses of tie2:EGFP+ (green) hearts stained immunohistochemically for myosin (red) show that treatment with 50 nM odanacatib restores normal morphology to MLII hearts and valves, as assayed by the number of MLII embryos with hearts that loop and valves that open and close (“clap”) at regular intervals following treatment. Percent values on images represent the number of embryos with the pictured phenotype. n = 100–125 embryos. Data are presented as mean ± SEM. **P < 0.01,***P < 0.001, ****P < 0.0001 using Dunnett’s test with correction (indicated by red box). Each dot represents the average of 20–25 embryos from 4–5 experiments. Scale bar: 25 μm. (C) In situ analyses of notch1b expression show restored differentiation of MLII valve cells (red arrowheads) following treatment with odanacatib. Percent values represent the number of embryos with the pictured phenotype. n = 75 embryos from 3 experiments. Scale bar: 50 μm. (D) Confocal analyses of embryos stained immunhistochemically for myosin (red) and pSmad1/5/8 (green) show odanacatib treatment increases BMP signaling in MLII embryos. Panels to the right represent higher-power images of boxed regions; white arrowheads denote nuclear localized pSmad1/5/8. Scale bar: 20 μm. (E) Graphs show percent cells containing Smad+ nuclei in WT, gnptab morphant, and odanacatib-treated embryos. n = 20–25 embryos imaged from 3 experiments. Data are presented as mean ± SEM. Student’s t test. **P < 0.01, ***P < 0.001. (F) Confocal images of myl7:RFP+ (labels cardiomyocytes red) and BRE:dsEGFP+ (green) embryos confirm odanacatib treatment improves BMP signaling in MLII embryos. Data quantified in Supplemental Figure 2. n = 10–15 embryos imaged from 2 experiments. Scale bar: 30 μm.