Flavin-containing monooxygenase 2 confers cardioprotection in ischemia models through its disulfide bond catalytic activity
Qingnian Liu, Jiniu Huang, Hao Ding, Yue Tao, Jinliang Nan, Changchen Xiao, Yingchao Wang, Rongrong Wu, Cheng Ni, Zhiwei Zhong, Wei Zhu, Jinghai Chen, Chenyun Zhang, Xiao He, Danyang Xiong, Xinyang Hu, Jian’an Wang
Qingnian Liu, Jiniu Huang, Hao Ding, Yue Tao, Jinliang Nan, Changchen Xiao, Yingchao Wang, Rongrong Wu, Cheng Ni, Zhiwei Zhong, Wei Zhu, Jinghai Chen, Chenyun Zhang, Xiao He, Danyang Xiong, Xinyang Hu, Jian’an Wang
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Research Article Cardiology

Flavin-containing monooxygenase 2 confers cardioprotection in ischemia models through its disulfide bond catalytic activity

Abstract

Myocardial infarction (MI) is characterized by massive cardiomyocyte (CM) death and cardiac dysfunction, and effective therapies to achieve cardioprotection are greatly needed. Here, we report that flavin-containing monooxygenase 2 (FMO2) levels were markedly increased in CMs in both ex vivo and in vivo models of ischemic injury. Genetic deletion of FMO2 resulted in reduced CM survival and enhanced cardiac dysfunction, whereas CM-specific FMO2 overexpression conferred a protective effect in infarcted rat hearts. Mechanistically, FMO2 inhibited the activation of ER stress–induced apoptotic proteins, including caspase 12 and C/EBP homologous protein (CHOP), by downregulating the unfolded protein response pathway. Furthermore, we identified FMO2 as a chaperone that catalyzes disulfide bond formation in unfolded and misfolded proteins through its GVSG motif. GVSG-mutated FMO2 failed to catalyze disulfide bond formation and lost its protection against ER stress and CM death. Finally, we demonstrated the protective effect of FMO2 in a human induced pluripotent stem cell–derived CM model. Collectively, this study highlights FMO2 as a key modulator of oxidative protein folding in CMs and underscores its therapeutic potential for treating ischemic heart disease.

Authors

Qingnian Liu, Jiniu Huang, Hao Ding, Yue Tao, Jinliang Nan, Changchen Xiao, Yingchao Wang, Rongrong Wu, Cheng Ni, Zhiwei Zhong, Wei Zhu, Jinghai Chen, Chenyun Zhang, Xiao He, Danyang Xiong, Xinyang Hu, Jian’an Wang

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Figure 7

FMO2-mediated cardiac protection against ER stress is dependent on catalysis of the disulfide bond.

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FMO2-mediated cardiac protection against ER stress is dependent on catal...
(A) Viability of differently treated CMs was detected with the Cell Counting Kit-8 (CCK-8), normalized to a parallel culture of the same cells that had not been exposed to DTT. (B) Contents of reduced thiols in hypoxia-exposed NRCMs transfected with LV-shFMO2 or LV-FMO2 lentivirus. Data shown for the differently treated groups were normalized to the hypoxia control. (C and D) The reaction of substrate GSH or peptide NRCSQGSCWN with FMO2. The GSSG/GSH ratio was measured, while the peptides was monitored according to fluorescence intensity (excitation 280 nm, emission 350 nm). (E and F) Images and analysis of TUNEL staining of hypoxia-exposed NRCMs treated or not with the disulfide bond–specific inhibitor TCEP. Scale bars: 100 μm. (G and H) Western blots and analysis showing alterations of GRP78 and ER stress–induced apoptotic proteins in hypoxia-exposed NRCMs with or without TCEP. (I) Contents of reduced thiols in hypoxia-exposed NRCMs transfected with PDI siRNA or LV-FMO2 lentivirus. Bar diagram of differently treated groups was normalized to the hypoxia control. (J and K) Western blots and analysis of protein expression of GRP78 and ER stress–induced apoptotic proteins in hypoxia-exposed NRCMs with PDI siRNA and LV-FMO2-FLAG lentivirus. Graphs summarize data from 3 independent experiments. Data are presented as the mean ± SEM. Comparisons between 2 groups were assessed by 2-tailed Student’s t test, comparisons among 3 or more groups were evaluated by 1-way ANOVA with Tukey’s test, and comparisons among groups after multiple treatments were evaluated by 2-way ANOVA with Tukey’s test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.