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Investigation of a dilated cardiomyopathy–associated variant in BAG3 using genome-edited iPSC-derived cardiomyocytes
Chris McDermott-Roe, … , Aron Geurts, Kiran Musunuru
Chris McDermott-Roe, … , Aron Geurts, Kiran Musunuru
Published November 14, 2019
Citation Information: JCI Insight. 2019;4(22):e128799. https://doi.org/10.1172/jci.insight.128799.
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Research Article Cardiology Genetics

Investigation of a dilated cardiomyopathy–associated variant in BAG3 using genome-edited iPSC-derived cardiomyocytes

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Abstract

Mutations in B cell lymphoma 2–associated athanogene 3 (BAG3) are recurrently associated with dilated cardiomyopathy (DCM) and muscular dystrophy. Using isogenic genome-edited human induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs), we examined how a DCM-causing BAG3 mutation (R477H), as well as complete loss of BAG3 (KO), impacts myofibrillar organization and chaperone networks. Although unchanged at baseline, fiber length and alignment declined markedly in R477H and KO iPSC-CMs following proteasome inhibition. RNA sequencing revealed extensive baseline changes in chaperone- and stress response protein–encoding genes, and protein levels of key BAG3 binding partners were perturbed. Molecular dynamics simulations of the BAG3-HSC70 complex predicted a partial disengagement by the R477H mutation. In line with this, BAG3-R477H bound less HSC70 than BAG3-WT in coimmunoprecipitation assays. Finally, myofibrillar disarray triggered by proteasome inhibition in R477H cells was mitigated by overexpression of the stress response protein heat shock factor 1 (HSF1). These studies reveal the importance of BAG3 in coordinating protein quality control subsystem usage within the cardiomyocyte and suggest that augmenting HSF1 activity might be beneficial as a means to mitigate proteostatic stress in the context of BAG3-associated DCM.

Authors

Chris McDermott-Roe, Wenjian Lv, Tania Maximova, Shogo Wada, John Bukowy, Maribel Marquez, Shuping Lai, Amarda Shehu, Ivor Benjamin, Aron Geurts, Kiran Musunuru

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Figure 1

Production and preliminary analysis of BAG3-R477H and BAG3-KO induced pluripotent stem cell (iPSC)-derived cardiomyocytes.

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Production and preliminary analysis of BAG3-R477H and BAG3-KO induced pl...
(A) Schematic representation of the BAG3 gene, WT genomic DNA (gDNA) sequence, and the central part of the single-stranded oligonucleotide (ssODN) sequence used to introduce the c.1430G>A (R477H) mutation, which causes DCM in humans (3), into iPSCs. (B) Sanger sequence traces and corresponding amino acid sequences of an unedited iPSC line (BAG3-WT, left) and an iPSC line heterozygous for the c.1430G>A (BAG3-RH) mutation (right). In A and B, underlined/bolded and italicized nucleotides denote the variant of interest and synonymous Cas9-blocking mutations, respectively. (C) BAG3 localization in BAG3-WT (WT), BAG3-R477H (RH), and BAG3-KO (KO) iPSC–derived cardiomyocytes. Green, BAG3; blue, DAPI. Scale bar: 20 μm. (D) Visualization of myofibrillar organization in BAG3-WT (WT), BAG3-R477H (RH), and BAG3-KO (KO) iPSC–derived cardiomyocytes. Red, cardiac troponin T; green, α-actinin; blue, DAPI. Scale bar: 20 μm. Data are representative of 3 independent experiments.

Copyright © 2021 American Society for Clinical Investigation
ISSN 2379-3708

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