Targeting E3 Ubiquitin Ligase Hrd1 Prevents Myocardial Ischemia-Reperfusion Injury Through Enhancing ALDH2 Enzymatic Activity

Abstract

BACKGROUND: Myocardial ischemia-reperfusion (I/R) injury presents a significant clinical challenge characterized by a complex pathological mechanism. The role of protein ubiquitination in I/R injury has not been systematically investigated. Global ubiquitinome profiling was conducted to identify the potential key players in myocardial I/R injury. METHODS: The ubiquitination levels of proteins in mouse hearts subjected to either sham surgery or I/R injury were analyzed using ubiquitinome. A combined analysis of ubiquitinome, single-cell RNA sequencing (RNA-seq), and proteomics data was employed to predict potential E3 ubiquitin ligases associated with myocardial I/R injury. Global heterozygous 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase degradation 1 (Hrd1) knockout, endothelial cell (EC)–specific Hrd1 deficiency (Hrd1 f/f ; Cdh5 Cre ), and EC-specific Hrd1 overexpression (AAV-EC-Hrd1) mice were used to assess the role of Hrd1 in myocardial I/R injury. Mass spectrometry and immunoprecipitation were used to elucidate the interaction between Hrd1 and aldehyde dehydrogenase 2 (ALDH2). Additionally, we assessed ubiquitination and vasomotor reactivity to clarify the mechanisms by which Hrd1 regulates ALDH2 activity and EC dysfunction during I/R injury. RESULTS: Ubiquitinome analysis revealed that protein ubiquitination exacerbates endothelial dysfunction after myocardial I/R injury. Integrative analysis of the ubiquitinome, proteomics, and single-cell RNA-seq revealed a significant upregulation of the E3 ubiquitin-protein ligase Hrd1 in CD45 + ECs. In both humans and mice, the level of endothelial Hrd1 protein was found to increase in response to I/R in vivo. Genetic ablation of Hrd1 significantly alleviated myocardial infarction, endothelial dysfunction, and infiltration of inflammatory cells after I/R injury. Mechanistically, Hrd1 promoted the K33-linked polyubiquitination of ALDH2 and then inhibited the formation of its active tetramers, which reduced the apoptosis of CD45 + ECs and exacerbated endothelial dysfunction through the NO/cGMP/PKG (nitric oxide–cyclic guanosine monophosphate–protein kinase G) signaling pathway. Furthermore, our findings demonstrated that pharmacological inhibition of Hrd1 robustly ameliorated myocardial I/R injury and endothelial dysfunction. CONCLUSIONS: Our findings demonstrated a previously unidentified crucial role of cardiac EC Hrd1 in myocardial I/R injury. Hrd1 may serve as a therapeutic target for preventing myocardial I/R injury.