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

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.