Nap1L1 Ubiquitination Degradation-dependent Protective Effects of Wnt2 on Cardiac Ischemia/Reperfusion Injury
Authors: Ying W , Liming C , Jinyi L I N , Chenxing H , Kejia J I N , Hao W , Jianguo J I A , Zhiwen D , Junbo G E , Hui G , Yunzeng Z O U 
Published: October 2024

Abstract

Background
Cardiomyocyte death during ischemia/reperfusion (I/R) injury occurs following the restoration of blood flow for obstructed coronary arteries, posing a challenge to timely reperfusion strategy for ischemic heart disease. This study aimed to investigate the potential protective role of Wnt2 against cardiomyocyte death after I/R injury.

Methods
Serum Wnt2 levels in patients with acute myocardial infarction (AMI) before and after percutaneous coronary intervention (PCI) and in mice subjected to I/R were measured by ELISA. Cardiomyocyte death, including apoptosis and ferroptosis, which were examined by TUNEL and thiobarbituric acid reactive substances (TBARs) assay, respectively. TMT6-based proteomics analysis was used to identify critical molecules mediating the effects of Wnt2.

Results
Serum Wnt2 level decreased after PCI in patients with MI and was negatively correlated with cTNT and CK-MB levels within the first 48 hours post-PCI. Wnt2 also decreased in I/R mice in compared with sham group.Supplement of rbWnt2 ameliorated I/R injury as characterized by the improved cardiac function and decreased infarct area and level of asynchronicity. Proteomics analysis KEGG Enrichment top 20 of differentially expressed proteins (DEPs) included reactive oxygen species (ROS). RbWnt2 inhibited cardiomyocytes apoptosis and ferroptosis by suppressed ROS level following I/R in vivo and in vitro. Based on proteomics analysis data, most of differentially expressed proteins (DEGs) were enriched in cytoplasm or nucleus. Among them, Nucleosome assembly protein-like 1(Nap1L1) was progressively increased in hearts following I/R, and showed a negative correlation with cardiac Wnt2 level during the I/R procedure. RbWnt2 treatment significantly attenuated the upregulation of Nap1L1 induced by I/R injury in vivo and in vitro. Hypoxia/Normoxia injury elicited Nap1L1 increased both in nucleus and cytoplasm, which was attenuated by rbWnt2 treatment in AMCMs. AAV9 mediated knockdown of Nap1l1 protects heart from apoptosis and ferroptosis while overexpression of Nap1l1 partly abolished the protective effects of Wnt2 in I/R mice. Mechanistically, Wnt2 upregulated anti-oxidative enzymes (Gpx1, Gpx4, Ucp3, Sod1, Sod2) by regulation of Nap1l1 to suppress ROS level and protect cardiomycoytes following I/R injury. Further analysis revealed that binding of Wnt2 and Lrp6 enhanced the expression of Tripartite motif 11 (TRIM11, a ubiquitin E3 ligase) and subsequently promoted the degradation of Nap1l1 in cardiomyocytes, which contributes to cardiac protection following I/R injury.

Conclusions
Wnt2 protects the heart against I/R-induced injury by inducing Nap1L1 degradation through Lrp6/Trim11 signaling, which was mediated by suppressing ROS levels via enhancing anti-oxidation enzymes to inhibits cardiomyocytes apoptosis and ferroptosis. These findings may offer new insights for developing therapeutic strategies to prevent I/R injury.Abstract GraphConclusion