UCHL1 protects against ischemic heart injury via activating HIF-1α signal pathway

Abstract

Ubiquitin carboxyl-terminal esterase L1 (UCHL1) has been thought to be a neuron specific protein and shown to play critical roles in Parkinson's Disease and stroke via de-ubiquiting and stabilizing key pathological proteins, such as α-synuclein. In the present study, we found that UCHL1 was significantly increased in both mouse and human cardiomyocytes following myocardial infarction (MI). When LDN-57444, a pharmacological inhibitor of UCHL1, was used to treat mice subjected to MI surgery, we found that administration of LDN-57444 compromised cardiac function when compared with vehicle treated hearts, suggesting a potential protective role of UCHL1 in response to MI. When UCHL1 was knockout by CRISPR/Cas 9 gene editing technique in human induced pluripotent stem cells (hiPSCs), we found that cardiomyocytes derived from UCHL1-/- hiPSCs were more susceptible to hypoxia/re-oxygenation induced injury as compared to wild type cardiomyocytes. To study the potential targets of UCHL1, a BioID based proximity labeling approach followed by mass spectrum analysis was performed. The result suggested that UCHL1 could bind to and stabilize HIF-1α following MI. Indeed, expression of HIF-1α was lower in UCHL1-/- cells as determined by Western blotting and HIF-1α target genes were also suppressed in UCHL1-/- cells as quantified by real time RT-PCR. Recombinant UCHL1 (rUCHL1) protein was purified by E. Coli fermentation and intraperitoneally (I.P.) delivered to mice. We found that administration of rUCHL1 could significantly preserve cardiac function following MI as compared to control group. Finally, adeno associated virus mediated cardiac specific UCHL1 delivery (AAV9-cTNT-m-UCHL1) was performed in neonatal mice. UCHL1 overexpressing hearts were more resistant to MI injury as compare to the hearts infected with control virus. In summary, our data revealed a novel protective role of UCHL1 on MI via stabilizing HIF-1α and promoting HIF-1α signaling.

Keywords: Deubiquitylating enzyme; HIF-1α; Ischemic cardiac injury; UCHL1.

Fig. 1

UCHL1 is upregulated in both human heart failure samples and murine myocardial infarction model. (A) Frozen human heart sections were immunohistochemically stained with UCHL1 antibody. (B) Quantification of UCHL1 expression in frozen human heart sections. (C) Western blotting (WB) analysis shows UCHL1 upregulation in infarct zone of mouse hearts, but not in the remote zone after MI surgery. (D) WB analysis for UCHL1 at different time pionts (3, 4, 7, 14, 28, 42 days) in infarct zone of mouse hearts after MI surgery. (E) Real-time RT-PCR analysis of UCHL1 mRNA levels in infarct heart tissues. (F) The quantitative densitometric analysis of UCHL1 protein level showing in panel D. (G) Immunofluorescence detection of UCHL1 in mouse hearts, UCHL1 immunofluorescence is rendered in red and α-sarcomeric actin (Alpha Sr-1) immunofluorescence in green (*P < 0.05, **P < 0.01, ***P < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Pharmacological inhibition of UCHL1 compromises cardiac function following MI. (A) The flow chart demonstrates the administration of LDN57444 to MI injured mice. (B) Representative images of M-mode echocardiography in different groups. (C) Quantification of ejection fraction (EF) after echocardiography of mice. (D) Representative images of Sirius Red staining of heart tissues. (E) Quantification analysis of fibrosis area in mouse hearts. (**P < 0.01). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

HIF-1α is a potential target of UCHL1 after myocardial infarction (MI). (A) Schematic representation of UCHL1 proximity-labeling by BioID. In the presence of biotin, the BirA-UCHL1 fusion protein biotinylates nearby proteins which can be affinity captured through streptavidin magnetic beads. (B) WB analysis of affinity captured material from BioID assay using cell lysates from HEK293T cells treated with hypoxia or CoCl₂. (C) WT and UCHL1−/− hiPSC-CM cells were treated with hypoxia for 0, 3, 6, 12 h, and the levels of HIF-1α and UCHL1 protein were analyzed by WB. (D) Quantification results from three independent WB of HIF-1α in panel D. (E) Quantification results from three independent western blots of UCHL1 in panel D. (F) Representative WB analysis of WT and UCHL1−/− hiPSC-CMs treated hypoxia with or without MG132. (G) Ubiquitination of HIF-1α was examined by immunoprecipitation (IP) analysis. The anti–HIF–1α antibody was used for IP, and ubiquitinated HIF-1α was detected by anti-ubiquitin antibody (*P < 0.05).

Fig. 4

UCHL1 controls nuclear translocation of HIF-1α. (A) hiPSC-CMs were subjected to Immunofluorescence staining of UCHL1 (red) and HIF-1α (green) in normoxic and hypoxic conditions. (B) Quantitation of HIF-1α influx in nuclei in WT and UCHL1−/− hiPSC-CMs in normoxic and hypoxic conditions. (C) Real time RT-PCR was performed to measure mRNA levels of UCHL1, HIF-1α target genes (CD39, LDHA, VEGF-α, PDK1 and BNIP3) in WT and UCHL1−/− hiPSC-CMs under normoxic and hypoxic conditions. (*P < 0.05, **P < 0.01, ***P < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

UCHL1 might be a therapeutic target to treat MI. (A) The flow chart of administration of rhUCHL1 to treat mice after MI surgery. (B) Representative images of M-mode echocardiography in different groups. (C) Quantification of ejection fraction (EF) after echocardiography of mice. (D) The flow chart of studying the role of AAV mediated cardiac specific delivery of UCHL1 in MI injured mice. (E) Representative images of M-mode echocardiography in different groups. (F) Quantification of ejection fraction (EF) after echocardiography of mice (*P < 0.05, **P < 0.01, ***P < 0.001).

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