Dysregulated autophagic flux plays a critical role in myocardial ischemia-reperfusion injury (MIRI), complicating cardiac reperfusion therapy. In this study, we identified OTUD4 as a potential regulator of autophagic flux in MIRI using CRISPR/Cas9 sgRNA sequencing. However, the underlying mechanism is poorly understood. The purpose of this study is to investigate the effects of OTUD4 on autophagic flux in OGD-R treated AC16 cells (IRI model in vitro) and LAD artery ligation induced myocardial ischemia-reperfusion mice (MIRI model in vivo). In the in vitro IRI cell model, OTUD4 knockdown significantly reversed impaired autophagic flux, increased mitochondrial membrane potential, and decreased LDH activity, ROS production, autophagy and apoptosis. Overexpression of OTUD4 showed the opposite result. In the in vivo MIRI model, OTUD4 knockdown also significantly decreased infarct area, improved cardiac structure and function, reduced serum BNP and LDH levels, attenuated cardiac tissue injury/fibrosis/myocardial hypertrophy, and ultimately exerted myocardial protective effects against ischemia-reperfusion injury. Importantly, OTUD4 knockdown inhibited autophagosome-associated markers (LC3II/LC3I, Beclin1, ATG9), autophagy substrate p62, increased lysosomal activity marker LAMP2, and activated the autophagy pathway (AKT/mTOR), thereby promoting the recovery of impaired autophagic flux in the MIRI model. Moreover, OTUD4 showed strong interaction with UBAC1, and OTUD4 deficiency decreases UBAC1 protein expression by impairing its deubiquitination, thereby regulating autophagy. In short, blocking OTUD4 restored damaged autophagic flux in I/R induced myocardial injury both in vivo and in vitro, inhibited myocardial cell apoptosis, and greatly improved cardiac function in ischemia-reperfusion mice. KEY MESSAGES: OTUD4 was identified as a key negative regulator of autophagy flux in myocardial ischemia-reperfusion injury (MIRI) via genome-wide CRISPR/Cas9 screening. OTUD4 knockdown exerts cardioprotective effects by reducing apoptosis and ROS generation and improving heart function in both in vitro and in vivo models. The interaction between OTUD4 and UBAC1 was confirmed, and OTUD4 maintains UBAC1 stability through deubiquitination, providing new insights into the ubiquitination regulatory mechanism in myocardial injury. Targeting OTUD4 has therapeutic potential for MIRI, as OTUD4 knockdown alleviated MIRI in both in vitro and in vivo models, suggesting the possibility of developing OTUD4 inhibitors for cardiac reperfusion treatment.
