UBA1 Mitigates Myocardial Ischemia/Reperfusion Injury by Attenuating Endoplasmic Reticulum-Mitochondria Contacts via Pdzd8 ubiquitination

BACKGROUND Myocardial ischemia/reperfusion injury (I/RI) represents a serious clinical complication in patients after acute myocardial infarction. Ubiquitin-activating enzyme 1 (UBA1) catalyzes the initial step of ubiquitination and plays a fundamental role in regulating protein homeostasis and related diseases. This study aims to elucidate the functional contribution of UBA1 to the pathogenesis of myocardial I/RI and to uncover its underlying mechanisms.

Single-cell RNA sequencing was employed to characterize UBA1 expression in human ischemic heart tissues. Myocardial I/R injury was examined in myocardial-specific UBA1 knockout (UBA1cko) mice, UBA1-overexpressing mice (rAAV9-UBA1), and corresponding controls. Neonatal rat cardiomyocytes underwent hypoxia/reoxygenation in vitro. Cardiac function and infarction were evaluated by echocardiography and pathological staining. Protein–protein interactions were analyzed via immunoprecipitation combined with mass spectrometry. The endoplasmic reticulum–mitochondrial contact sites (ERMCSs) and mitochondrial ultrastructure were evaluated through transmission electron microscopy and confocal imaging.

UBA1 expression was significantly downregulated in human and murine ischemic myocardium, especially in cardiomyocytes. UBA1cko mice exhibited aggravated I/RI with greater infarct size, impaired function, apoptosis, elevated intracellular Ca2+ levels, mitochondrial dysfunction, and ER stress, whereas UBA1 overexpression conferred cardioprotective effects. Mechanistically, UBA1 directly bound to and ubiquitinated Pdzd8, a key ERMCS-tethering protein, thereby promoting its degradation, which inhibited ERMCS formation and improved mitochondrial dysfunction and ER stress. Moreover, knockdown of Pdzd8 via rAAV9-siRNA effectively mitigated UBA1 knockout-induced myocardial damage. Additionally, administration of auranofin (AF), a U.S. Food and Drug Administration-approved drug for treating rheumatoid arthritis, markedly alleviated myocardial I/RI via activating UBA1 in vivo and in vitro.

UBA1 confers protection against myocardial I/RI by limiting ERMCS formation through Pdzd8 ubiquitination. Activating UBA1 or targeting Pdzd8 as a potential therapeutic strategy for the treatment of ischemic heart disease. GRAPHIC ABSTRACT A graphic abstract is available for this article. What Is New? UBA1 expression is downregulated in human and murine ischemic myocardium, especially in cardiomyocytes. Cardiac deletion of UBA1 significantly exacerbates myocardial ischemia/reperfusion injury (I/RI), whereas cardiac UBA1 overexpression confers a marked protective effect. UBA1 interacts with Pdzd8 (PDZ domain containing 8) and facilitates its ubiquitination and subsequent degradation, which then reduces endoplasmic reticulum-mitochondria contact sites (ERMCSs) and ameliorates mitochondrial dysfunction and ER stress, protecting myocardial I/RI. Pharmacological activation of UBA1 with the FDA-approved drug auranofin attenuates myocardial I/R injury and improves heart dysfunction. What Are the Clinical Implications? UBA1 represents a new therapeutic target for myocardial I/RI. Activating UBA1 or targeting Pdzd8 may offer a promising therapeutic strategy for mitigating myocardial I/RI and heart failure, underscoring its potential for clinical translation. Competing Interest Statement The authors have declared no competing interest. Nonstandard Abbreviations and Acronyms - AAV9 - adeno-associated virus serotype 9 - AF - Auranofin - CKO - cardiomyocyte-specific UBA1 knockout - Co-IP - Co-immunoprecipitation - DEGs - differentially expressed genes - DUBs - deubiquitinases - ECHO - echocardiography - ERMCs - endoplasmic reticulum–mitochondria contacts - ERMCSs - endoplasmic reticulum-mitochondrial contact sites - EF - ejection fraction - FS - fractional shortening - H/R - hypoxia/reoxygenation - I/RI - ischemia reperfusion injury - LDH - lactate dehydrogenase - MAMs - mitochondria-associated ER membranes - MI - myocardial infarction - NRCFs - neonatal rat fibroblasts - NRCMs - neonatal rat cardiomyocytes - Pdzd8 - PDZ domain containing 8 - ROS - reactive oxygen species - TEM - transmission electron microscopy - TUNEL - TdT-mediated dUTP nick-end labeling - Ub - Ubiquitin - UBA1 - Ubiquitin-activating enzyme E1 - WT - wild type