Abstract 4368363: TRIM35 Exacerbates Myocardial Delayed Reperfusion Injury by Ubiquitin-Mediated Degradation of EN1 and Disruption of Mitochondrial Respiratory Complex

Abstract

Background: Delayed reperfusion (DR) in myocardial ischemia causes long-term cardiac dysfunction, partially due to impaired mitochondrial quality control. TRIM35 is an E3 ubiquitin ligase implicated in cellular stress responses and protein turnover. TRIM family proteins critically regulate mitochondrial homeostasis. Given the essential role of mitochondria in cardiomyocyte survival during DR injury, elucidating the specific role of TRIM35 in regulating mitochondrial function may yield novel therapeutic insights. Methods: We first assessed TRIM35 expression dynamics in mouse hearts subjected to I/R injury and in hypoxia/reoxygenation-treated cardiomyocytes. Cardiomyocyte-specific Trim35 knockout (Trim35 CKO) mice and TRIM35-overexpressing cardiomyocytes were used to evaluate its functional impact. Proteomic and biochemical assays, including co-immunoprecipitation and ubiquitination analysis, were conducted to explore downstream mechanisms and protein targets of TRIM35. Results: TRIM35 expression was significantly upregulated in both cardiac tissues and cardiomyocytes during the late reperfusion phase, especially at 3 to 7 days post-DR, suggesting its involvement in subacute injury responses. Trim35 CKO mice demonstrated significantly reduced infarct size, attenuated cardiomyocyte apoptosis, and substantially preserved systolic function. Additionally, Trim35 deficiency preserved mitochondrial membrane potential, enhanced the expression of mitochondrial respiratory complexes and promoted ATP production. In contrast, overexpression of TRIM35 diminished respiratory complex expression and exacerbated mitochondrial damage. Mechanistically, TRIM35 was found to promote K48-linked ubiquitination of the transcription factor EN1, facilitating its proteasomal degradation. Loss of TRIM35 stabilized EN1 protein levels, thereby enhancing PGC-1α/TFAM-mediated transcription of mitochondrial respiratory genes. Critically, EN1 overexpression rescued the detrimental effects of TRIM35 overexpression on mitochondrial respiratory complex expression and structural integrity. Conclusions: TRIM35 promotes the degradation of EN1 via K48-linked ubiquitination, contributing to impaired mitochondrial function and exacerbated myocardial DR injury. Targeting TRIM35 may enhance EN1 stability and represent a novel strategy to preserve mitochondrial homeostasis and protect the heart from ischemic injury.