Peptidylarginine deiminase 4 deficiency alleviates hypoxia/reoxygenation-induced cardiomyocyte injury

Abstract

Background Cardiac ischemia reperfusion (I/R) injury is a serious consequence of reperfusion therapy for myocardial infarction (MI). Peptidylarginine deiminase 4 (PAD4) is a calcium-dependent enzyme that catalyzes the citrullination of proteins. In previous studies, PAD4 inhibition protected distinct organs from I/R injury by preventing the formation of neutrophil extracellular traps (NETs) and attenuating inflammatory responses. Here, we hypothesized that cardiomyocyte PAD4 expression may play a role in acute I/R injury. Methods Infarct size was determined in isolated pressure constant-perfused hearts from WT and PAD4-deficient (PAD4-/-) mice. Additionally, extracellular reactive oxygen species (ROS) and cell viability were quantified in freshly isolated adult cardiomyocytes exposed to hypoxia followed by reoxygenation (H/R). Resistance to oxidative stress was proven in both genotypes by treatment of neonatal cardiomyocytes with hydrogen peroxide. Moreover, intracellular ROS formation, ATP production, mitochondrial membrane polarisation, caspase-3 activation, and cell viability were quantified after hypoxia followed by 4 h and 20 h of reoxygenation, respectively. The PAD4-specific inhibitor GSK484 was added before H/R or at reperfusion in certain experiments. Results Infarct size was smaller in PAD4-/- hearts following I/R when compared to the WT. Similarly, the viability of adult and neonatal PAD4-/- cardiomyocytes was better preserved after H/R, accompanied by reduced ROS formation. PAD4 deficiency maintained mitochondrial integrity and protected neonatal cardiomyocytes against apoptosis. However, these cells did not exhibit resistance to hydrogen peroxide-induced cell death, indicating an unaltered antioxidative state. Whereas pharmacological PAD4 inhibition by GSK484 before H/R sustained intracellular ATP levels in WT cardiomyocytes, administration of GSK484 at reoxygenation did not. However, GSK484 significantly improved cardiomyocyte metabolic activity, regardless of the time of administration. Conclusions Our study is the first to demonstrate that PAD4 expression in cardiomyocytes contributes to H/R injury independent of systemic immune responses and NETs. Consequently, PAD4 may serve as a therapeutic target to alleviate I/R injury.