Progress Report Year 2 MPS/ISMRD Grant to Heather Flanagan-Steet – Investigating the Role ofCathepsin Proteases in MLII Cardiac Pathology
In the last year we have made progress (detailed below) on several aspects of the aims outlined in thisgrant. In addition to progress on the proposed studies, we have made two important advances that will
have a major impact on our ongoing pursuit of molecular mediators of MLII cardiac pathology. Theseinclude 1) the generation of five unique TALEN-mediated MLII mutant lines and 2) the isolation of aseparate MLII mutant line from a sperm TILLING screen. During this year we not only established pure –outcrossed stable lines, but we also confirmed the genetic and biochemical characteristics of these lines.
These animals are essential to confirm all of our morpholino-based findings, for analyses of later stage
aspects of disease, and for small molecule screens. Importantly, several of the lines exhibit the same
craniofacial and cardiac anomalies noted in morpholino-generated animals. When published we will
credit MPS/ISMRD for contributing to the establishment of these vital tools.
Aim 1: Assess individual contribution of cathepsin K and L toward cardiac defects in ML zebrafish.
1) Cathepsin expression and generation of transgenic animals. Immunohistochemical analyses ofcathepsin K and L demonstrate that both proteases are expressed in multiple heart tissues, including themyocardium, endocardium, and epicardium. These analyses have guided the choice of tissue specificpromoters for transgenic constructs that will be used to generate zebrafish expressing fluorophor tagged
WT and mutant versions of Cts K and L. Toward this goal, we have now successfully shown that followingmRNA injection, the CtsK-mCherry fusion protein is both expressed and active in developing embryos.
Establishing these parameters was critical before proceeding with transgenesis. These animals, as well as
multiple ctsk variants/mutants, particularly those that mimick MLII, are in production.
2) Inhibition of cathespin K in MLII. To assess CtsK’s contribution toward ML cardiac pathology, its
expression or activity was inhibited in the MLII background. CtsK expression was genetically inhibitedusing one of two gene specific morpholinos and its activity reduced pharmacologically using the FDAapproved cathepsin K inhibitor Odanacatib. All aspects of cardiac morphology and function, including
formation of the AV valve, unidirectional blood flow, and heart contraction were substantially improvedby these treatments.
Aim 2 – Determine whether increased cathepsin activity impacts TGFß signaling in MLII hearts.
1) Are there differences in either TGFß or BMP signaling in WT and MLII hearts? In combination with TGFßand BMP-reporter transgenic animals, immunohistochemical analyses of pSmad levels was used to assessdifferences in signaling between WT and MLII hearts. These analyses revealed important changes inthese pathways that were restored by inhibition of cathepsin K.
Aim 3 – Determine which patient mutations are pathogenic for cardiac dysfunction.
To identify which patient mutations are associated with altered cardiac morphology and function, we
have introduced mRNA bearing specific lesions in the morphant background and compared their ability
to rescue heart phenotypes with ML animals co-injected with WT mRNA. We have analyzed threedifferent sets of mutations including several within the DMAP domain (including K732N), threemutations within Notch domain 1 (C442Y, C461G, and C468S), and one mutation in Notch domain 2. Thus
far analyses have been limited to gross morphology. Unlike WT gnptab mRNA, which reduces cardiac
edema, restores normal cardiac morphology, and increases blood flow in ≥85% of the animals analyzed,
mRNA bearing the K732N mutation did not significantly restore any of the tested parameters. Therefore,
as previously noted in the cartilage, the DMAP domain is essential for normal phosphotransferase
function – and mutations within it are highly likely to be pathogenic in both the cartilage/bone and theheart. In contrast, preliminary analyses of the three Notch domain 1 mutations suggest that the C442Y
lesion, which has been associated with MLIII, is less pathogenic in the heart than the ML-intermediate
lesion C468S. This is supported by the fact that mRNA“rescue” experiments show improved cardiac
morphology and reduced edema in 85% of the MLII animals injected with the C442Y-containing mRNA
versus only 40% recovery with the mRNA carrying the C468S lesion. mRNAs bearing either the C461G(Notch 1/MLIII) or C505Y (Notch2/MLIII) rescued gross cardiac pathology ~60% of the time, suggestingthat in addition to differences in pathogenic severity between MLII and MLIII, different mutations within
a single ML class may have different degrees of pathogenicity. More in depth experiments are currently
underway. Collectively these data support the idea that genotype-phenotype correlations can beestablished in this system, and as such may eventually combine with patient data to predict the severity
and course of mutation specific tissue pathologies.
Summary of progress to date:
Although complete understanding of the complicated nature of MLII cardiac pathology still demands
more work, we are highly encouraged by the findings to date. First, the fact that both cathepsin K
knockdown and Ctsk inhibitors improved MLII heart development is very promising. These data not only
support a central role for cathepsin(s) in primary pathogenesis but point to FDA-approved drugs for
future consideration. Second, the fact that both cathepsin K and TGFß signaling are involved in cardiac
pathology suggests common mechanisms underlie ML skeletal and heart dysfunction. If true, this would
not only be an important advance for future scientific investigation, but also mean that an individual
modality may therapeutically improve both systems. Third, the tools generated thus far and thoseunderway will serve as novel platforms to both further investigate these and other emerging mechanisms
and to rapidly screen the efficacy of potential drugs.