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Patient mutations linked to arrhythmogenic cardiomyopathy enhance calpain-mediated desmoplakin degradation
Ronald Ng, … , Maegen A. Ackermann, Stuart G. Campbell
Ronald Ng, … , Maegen A. Ackermann, Stuart G. Campbell
Published June 13, 2019
Citation Information: JCI Insight. 2019;4(14):e128643. https://doi.org/10.1172/jci.insight.128643.
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Research Article Cardiology Genetics

Patient mutations linked to arrhythmogenic cardiomyopathy enhance calpain-mediated desmoplakin degradation

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Abstract

Arrhythmogenic cardiomyopathy (ACM) is an inherited disorder with variable genetic etiologies. Here, we focused on understanding the precise molecular pathology of a single clinical variant in DSP, the gene encoding desmoplakin. We initially identified a potentially novel missense desmoplakin variant (p.R451G) in a patient diagnosed with biventricular ACM. An extensive single-family ACM cohort was assembled, revealing a pattern of coinheritance for R451G desmoplakin and the ACM phenotype. An in vitro model system using patient-derived induced pluripotent stem cell lines showed depressed levels of desmoplakin in the absence of abnormal electrical propagation. Molecular dynamics simulations of desmoplakin R451G revealed no overt structural changes, but a significant loss of intramolecular interactions surrounding a putative calpain target site was observed. Protein degradation assays of recombinant desmoplakin R451G confirmed increased calpain vulnerability. In silico screening identified a subset of 3 additional ACM-linked desmoplakin missense mutations with apparent enhanced calpain susceptibility, predictions that were confirmed experimentally. Similar to R451G, these mutations are found in families with biventricular ACM. We conclude that augmented calpain-mediated degradation of desmoplakin represents a shared pathological mechanism for select ACM-linked missense variants. This approach for identifying variants with shared molecular pathologies may represent a powerful new strategy for understanding and treating inherited cardiomyopathies.

Authors

Ronald Ng, Heather Manring, Nikolaos Papoutsidakis, Taylor Albertelli, Nicole Tsai, Claudia J. See, Xia Li, Jinkyu Park, Tyler L. Stevens, Prameela J. Bobbili, Muhammad Riaz, Yongming Ren, Christopher E. Stoddard, Paul M.L. Janssen, T. Jared Bunch, Stephen P. Hall, Ying-Chun Lo, Daniel L. Jacoby, Yibing Qyang, Nathan Wright, Maegen A. Ackermann, Stuart G. Campbell

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Figure 9

Increased calpain degradation is common to other ACM-linked mutations in DSP.

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Increased calpain degradation is common to other ACM-linked mutations in...
(A) Model showing the location of 7 other ACM-linked desmoplakin mutations in relation to the R451G variant. Together, these mutations constitute a potential hotspot with shared molecular pathology. (B) Molecular dynamic simulations were run on these 7 additional ACM-linked mutations, and the number of intramolecular interactions around the calpain target site were calculated. Histograms showing the frequency of intramolecular interactions reveal a variety of mutation-associated changes, including reduced interactions for 3 mutants in particular (S299R, S442F, and S507F). (C) Models of desmoplakin showing the surface exposure of the affected calpain site (red) suggest that exposure in S299R desmoplakin is greater than for the WT structure. This predicted increase was statistically significant when examined across several repeat simulations (*P < 0.05, 2-tailed t test, n = 9 WT and n = 4 S299R). (D) Each of the 7 hotspot mutations was recombinantly expressed in DSP NH2-terminal fragments, purified, and incubated with calpain and Ca2+ for 30 minutes. Total protein remaining was determined for each mutation. S299R, S442F, and S507F each showed significant degradation relative to WT fragment over the same interval (*P < 0.05 for 1-way ANOVA with Tukey’s multiple comparisons test; WT n = 3; variants n = 3). Error bars represent SEM. (E) In order to test the predictive ability of the molecular dynamic simulations, regression analysis was performed on predicted calpain site surface area exposure and the percentage desmoplakin remaining after calpain treatment for each variant and WT. The 2 quantities were significantly correlated (R2 = 0.63, P < 0.05, linear regression analysis).

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