Inadequate ubiquitination-proteasome coupling contributes to myocardial ischemia-reperfusion injury
Chengjun Hu, … , Jinbao Liu, Xuejun Wang
Chengjun Hu, … , Jinbao Liu, Xuejun Wang
Published December 3, 2018; First published September 11, 2018
Citation Information: J Clin Invest. 2018;128(12):5294-5306. https://doi.org/10.1172/JCI98287.
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Categories: Research Article Cardiology

Inadequate ubiquitination-proteasome coupling contributes to myocardial ischemia-reperfusion injury

Abstract

The ubiquitin-proteasome system (UPS) degrades a protein molecule via 2 main steps: ubiquitination and proteasomal degradation. Extraproteasomal ubiquitin receptors are thought to couple the 2 steps, but this proposition has not been tested in vivo with vertebrates. More importantly, impaired UPS performance plays a major role in cardiac pathogenesis, including myocardial ischemia-reperfusion injury (IRI), but the molecular basis of UPS impairment remains poorly understood. Ubiquilin1 is a bona fide extraproteasomal ubiquitin receptor. Here, we report that mice with a cardiomyocyte-restricted knockout of Ubiquilin1 (Ubqln1-CKO mice) accumulated a surrogate UPS substrate (GFPdgn) and increased myocardial ubiquitinated proteins without altering proteasome activities, resulting in late-onset cardiomyopathy and a markedly shortened life span. When subject to regional myocardial ischemia-reperfusion, young Ubqln1-CKO mice showed substantially exacerbated cardiac malfunction and enlarged infarct size, and conversely, mice with transgenic Ubqln1 overexpression displayed attenuated IRI. Furthermore, Ubqln1 overexpression facilitated proteasomal degradation of oxidized proteins and the degradation of a UPS surrogate substrate in cultured cardiomyocytes without increasing autophagic flux. These findings demonstrate that Ubiquilin1 is essential to cardiac ubiquitination-proteasome coupling and that an inadequacy in the coupling represents a major pathogenic factor for myocardial IRI; therefore, strategies to strengthen coupling have the potential to reduce IRI.

Authors

Chengjun Hu, Yihao Tian, Hongxin Xu, Bo Pan, Erin M. Terpstra, Penglong Wu, Hongmin Wang, Faqian Li, Jinbao Liu, Xuejun Wang

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Figure 1

Ubqln1 colocalizes with the proteasome and is recruited to the ERAD pathway by ER stress in NRVMs.

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Ubqln1 colocalizes with the proteasome and is recruited to the ERAD path...
(A) Representative confocal micrographs of double-immunofluorescence staining for Ubqln1 (red) and Psmb5 (green). FLAG-tagged Ubqln1 was overexpressed in NRVMs grown on chamber slides through adenovirus-mediated gene delivery. The cells were fixed and processed for indirect immunofluorescence staining for FLAG and Psmb5. Scale bar: 5 μm. (B) Immunoblot analyses for the indicated proteins in the FLAG-Ubqln1 immunoprecipitates. Cultured NRVMs were infected with adenoviruses expressing FLAG-Ubqln1 or Ad–β-Gal 24 hours before treatment with tunicamycin (60 μg/ml) for 6 hours. The cell lysates were subject to co-IP with a mouse monoclonal anti-FLAG Ab. The IP products were fractionated with 10% SDS-PAGE and transferred to PVDF membrane for immunoblot. Loading control used in-gel stain-free total protein imaging (Supplemental Figure 1). (C and D) Western blot image (C) and densitometry data (D) of Western blot analysis for myocardial Ubqln1 in WT mice subjected to myocardial IRI (30 minutes/24 hours) or sham surgery. Both 1% Triton X-100 PBS-soluble and -insoluble fractions were examined. Tg, Ubqln1 Tg mouse myocardial tissue used as a positive control to validate the Ubqln1 Ab. Loading controls used in-gel stain-free total protein imaging (Supplemental Figure 2). Mean ± SD are superimposed. Two-tailed t test with Welch’s correction. For each group of all mouse experiments presented here and after, equal or nearly equal numbers of male and female mice were used. Each dot and each lane in Western blot image represents an individual animal.