Loss-of-function mutations in co-chaperone BAG3 destabilize small HSPs and cause cardiomyopathy
Xi Fang, Julius Bogomolovas, Tongbin Wu, Wei Zhang, Canzhao Liu, Jennifer Veevers, Matthew J. Stroud, Zhiyuan Zhang, Xiaolong Ma, Yongxin Mu, Dieu-Hung Lao, Nancy D. Dalton, Yusu Gu, Celine Wang, Michael Wang, Yan Liang, Stephan Lange, Kunfu Ouyang, Kirk L. Peterson, Sylvia M. Evans, Ju Chen
Xi Fang, Julius Bogomolovas, Tongbin Wu, Wei Zhang, Canzhao Liu, Jennifer Veevers, Matthew J. Stroud, Zhiyuan Zhang, Xiaolong Ma, Yongxin Mu, Dieu-Hung Lao, Nancy D. Dalton, Yusu Gu, Celine Wang, Michael Wang, Yan Liang, Stephan Lange, Kunfu Ouyang, Kirk L. Peterson, Sylvia M. Evans, Ju Chen
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Research Article Cardiology

Loss-of-function mutations in co-chaperone BAG3 destabilize small HSPs and cause cardiomyopathy

Abstract

Defective protein quality control (PQC) systems are implicated in multiple diseases. Molecular chaperones and co-chaperones play a central role in functioning PQC. Constant mechanical and metabolic stress in cardiomyocytes places great demand on the PQC system. Mutation and downregulation of the co-chaperone protein BCL-2–associated athanogene 3 (BAG3) are associated with cardiac myopathy and heart failure, and a BAG3 E455K mutation leads to dilated cardiomyopathy (DCM). However, the role of BAG3 in the heart and the mechanisms by which the E455K mutation leads to DCM remain obscure. Here, we found that cardiac-specific Bag3-KO and E455K-knockin mice developed DCM. Comparable phenotypes in the 2 mutants demonstrated that the E455K mutation resulted in loss of function. Further experiments revealed that the E455K mutation disrupted the interaction between BAG3 and HSP70. In both mutants, decreased levels of small heat shock proteins (sHSPs) were observed, and a subset of proteins required for cardiomyocyte function was enriched in the insoluble fraction. Together, these observations suggest that interaction between BAG3 and HSP70 is essential for BAG3 to stabilize sHSPs and maintain cardiomyocyte protein homeostasis. Our results provide insight into heart failure caused by defects in BAG3 pathways and suggest that increasing BAG3 protein levels may be of therapeutic benefit in heart failure.

Authors

Xi Fang, Julius Bogomolovas, Tongbin Wu, Wei Zhang, Canzhao Liu, Jennifer Veevers, Matthew J. Stroud, Zhiyuan Zhang, Xiaolong Ma, Yongxin Mu, Dieu-Hung Lao, Nancy D. Dalton, Yusu Gu, Celine Wang, Michael Wang, Yan Liang, Stephan Lange, Kunfu Ouyang, Kirk L. Peterson, Sylvia M. Evans, Ju Chen

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Figure 2

BAG3 deficiency leads to the downregulation of protein levels of sHSPs.

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BAG3 deficiency leads to the downregulation of protein levels of sHSPs. ...
(A–D) Representative immunoblots (A and C) and quantification analysis (B and D) of BAG3, HSP90, HSP70, HSC70 (A and B), and HSPB5, -6, -7, and -8 (C and D) in adult cardiomyocytes isolated from control and CKO mice. n = 4 mice per group. (E and F) Neonatal cardiomyocytes were isolated from Bag3fl/fl mice and infected with Ad-Cre (20 MOI) for 24, 48, and 72 hours. Ad-lacZ was used as a control. Representative immunoblots (E) and quantification analysis (F) of BAG3 and HSPB5, -6, and -8. n = 4. GAPDH served as a loading control. (G and H) Neonatal cardiomyocytes were isolated from Bag3 global-KO mice or WT controls and infected with BAG3-overexpressing adenovirus (Ad-BAG3) (20 MOI) for 36 hours. GFP adenovirus (Ad-GFP) was used as a control. Representative immunoblots (G) and quantification analysis (H) of HSPB5, -6, and -8. n = 4. GAPDH served as a loading control. Data are represented as the mean ± SEM. *P < 0.05, by 2-tailed Student’s t test or 2-way ANOVA.