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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 3

Engineered heart tissues from patient-derived induced pluripotent stem cells exhibit reduced levels of desmoplakin.

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Engineered heart tissues from patient-derived induced pluripotent stem c...
(A) Engineered heart tissues (EHTs) were created by differentiating cardiomyocytes from patient-derived induced pluripotent stem cells (iPSCs) and seeding cells into decellularized porcine myocardial slices. Constructs were used to assess contractility and electrical conduction velocity. (B) EHTs were generated using cardiomyocytes differentiated from G(+)P(+) patient III-36 and G(–)P(–) sibling III-35, denoted as EHTWT/R451G and EHTWT/WT, respectively. Bright-field images of EHTs show similar matrix compaction and morphology between the 2 types. Scale bar: 1 mm. (C) Representative immunoblot of desmoplakin (DSP) protein levels in EHTs shows substantial loss in the patient-derived tissue, which was statistically significant when quantified across several samples (*P < 0.05 for 2-tailed unpaired t test; n = 5 WT and n = 7 R451G). (D) Ca2+ transients collected from EHTWT/R451G and EHTWT/WT showed no differences between the 2 tissue types. (E) Measurements of longitudinal conduction velocity in EHTs showed no differences between EHTWT/R451G and EHTWT/WT (unpaired t test; n = 7 and 8 respectively). (F) Protein levels of connexin-43 (Cx43) and phosphorylated (Ser368) Cx43 in EHTs normalized to sarcomeric actin reveals significantly reduced Cx43 and significantly elevated levels of phosphorylated Cx43 in EHTWT/R451G (*P < 0.05 for 2-tailed unpaired t test, n = 3). (G) Representative force transients collected simultaneously with above Ca2+ transients reveal a delayed time to peak force in EHTWT/R451G. (H) Maximal force produced by EHTs. (I) Time from stimulus to maximal force. (J) Time elapsed from point of maximal force to 50% relaxation (RT50). ***P < 0.001 for 2-tailed unpaired t test with Bonferroni correction (n = 16 EHTWT/WT and n = 8 EHTWT/R451G). Error bars represent SEM.

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