A modifier screen identifies DNAJB6 as a cardiomyopathy susceptibility gene
Yonghe Ding, Pamela A. Long, J. Martijn Bos, Yu-Huan Shih, Xiao Ma, Rhianna S. Sundsbak, Jianhua Chen, Yiwen Jiang, Liqun Zhao, Xinyang Hu, Jianan Wang, Yongyong Shi, Michael J. Ackerman, Xueying Lin, Stephen C. Ekker, Margaret M. Redfield, Timothy M. Olson, Xiaolei Xu
Yonghe Ding, Pamela A. Long, J. Martijn Bos, Yu-Huan Shih, Xiao Ma, Rhianna S. Sundsbak, Jianhua Chen, Yiwen Jiang, Liqun Zhao, Xinyang Hu, Jianan Wang, Yongyong Shi, Michael J. Ackerman, Xueying Lin, Stephen C. Ekker, Margaret M. Redfield, Timothy M. Olson, Xiaolei Xu
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Research Article Cardiology Genetics

A modifier screen identifies DNAJB6 as a cardiomyopathy susceptibility gene

Abstract

Mutagenesis screening is a powerful forward genetic approach that has been successfully applied in lower-model organisms to discover genetic factors for biological processes. This phenotype-based approach has yet to be established in vertebrates for probing major human diseases, largely because of the complexity of colony management. Herein, we report a rapid strategy for identifying genetic modifiers of cardiomyopathy (CM). Based on the application of doxorubicin stress to zebrafish insertional cardiac (ZIC) mutants, we identified 4 candidate CM-modifying genes, of which 3 have been linked previously to CM. The long isoform of DnaJ (Hsp40) homolog, subfamily B, member 6b (dnajb6b(L)) was identified as a CM susceptibility gene, supported by identification of rare variants in its human ortholog DNAJB6 from CM patients. Mechanistic studies indicated that the deleterious, loss-of-function modifying effects of dnajb6b(L) can be ameliorated by inhibition of ER stress. In contrast, overexpression of dnajb6(L) exerts cardioprotective effects on both fish and mouse CM models. Together, our findings establish a mutagenesis screening strategy that is scalable for systematic identification of genetic modifiers of CM, feasible to suggest therapeutic targets, and expandable to other major human diseases.

Authors

Yonghe Ding, Pamela A. Long, J. Martijn Bos, Yu-Huan Shih, Xiao Ma, Rhianna S. Sundsbak, Jianhua Chen, Yiwen Jiang, Liqun Zhao, Xinyang Hu, Jianan Wang, Yongyong Shi, Michael J. Ackerman, Xueying Lin, Stephen C. Ekker, Margaret M. Redfield, Timothy M. Olson, Xiaolei Xu

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Figure 5

Overexpression of Dnajb6(L) in cardiomyocytes protected against DOX-induced cardiomyopathy in mouse.

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Overexpression of Dnajb6(L) in cardiomyocytes protected against DOX-indu...
(A) Schematics of adeno-associated virus serotype-9–Dnajb6(L) (AAV9-Dnajb6[L]) and doxorubicin (DOX) injection protocol in mice. (B) Western blot (upper) and quantification (lower) analysis of Dnajb6, glucose-regulated protein 78 (Grp78), and C/EBP homologous protein (CHOP) protein expression levels at 3 months after 5 × 1011 genomic copy of AAV9-Dnajb6(L) virus preinjection with or without injection of 12 mg/kg body weight of DOX. Values are expressed as relative unit to the GAPDH input. Data represent mean ±SEM. *P < 0.05, 1-way ANOVA comparisons. (C) Kaplan-Meier survival curves of mice preinjected with AAV9-Dnajb6(L) or PBS control and then injected with DOX. Overexpression of Dnajb6(L) through AAV9-mediated gene delivery protected against DOX-induced mice death. Data represent mean ±SEM. *P < 0.05, log-rank test. (D) Percent ejection fraction of mice at 3 months after DOX injection with or without AAV9-Dnajb6(L) preinjection. AAV9-Dnajb6(L) virus preinjected mice had preserved cardiac function. Data represent mean ±SEM. *P < 0.05, 2-way ANOVA comparisons. (E) H&E staining (upper panel) and transmission electron microscopic analysis (lower panel) of mouse myocardium at 3 months after DOX injection with or without AAV9-Dnajb6(L) preinjection. Arrows indicate cytoplasmic vacuoles. The images are representative of n = 3 per treatment. Scale bars: 100 μm (upper) and 2 μm (lower).