PAD2 disturbs cardiomyocyte calcium homeostasis by citrullinating SERCA2a protein in hemorrhagic shock induced arrhythmia

BACKGROUND: Malignant arrhythmia induced by traumatic hemorrhage is a leading cause of early mortality in hemorrhagic shock. Understanding the mechanisms driving these arrhythmias and identifying therapeutic targets are critical for improving early survival in patients with traumatic hemorrhagic shock. METHODS: Peripheral blood samples from patients with hemorrhagic shock were collected and analyzed for peptidylarginine deiminase 2 (PAD2) protein levels using ELISA. Pad2 knockout mice ( Pad2 -/- , Pad2 KO) were generated, and the hemorrhagic shock model was constructed via femoral artery cannulation and bloodletting. Cardiomyocytes were isolated and contractility and calcium content were measured by confocal microscopy. PAD2 subcellular localization was assessed through immunofluorescence and Western blotting. Proteins interacting with PAD2 in cardiomyocytes were identified using co-immunoprecipitation followed by mass spectrometry (CoIP-MS). The effect of PAD2 on sarcoplasmic reticulum calcium-ATPase 2a (SERCA2a) activity and citrullination was evaluated through enzyme activity assays and protein citrullination detection. AAV9-PAD2 was injected into mice via tail vein to induce in vivo overexpression of PAD2 in the myocardium. The effects of PAD2 enzymatic activity mutations and a PAD2-specific inhibitor on survival rate and arrhythmia following hemorrhagic shock were assessed through intraperitoneal injection. RESULTS: PAD2 protein levels were significantly elevated in the peripheral blood of patients with hemorrhagic shock. Pad2 knockout improved calcium homeostasis in the sarcoplasmic reticulum of cardiomyocytes and alleviated post-shock arrhythmia in mice. Following hypoxia, PAD2 exhibited increased colocalization with the sarcoplasmic reticulum. During hypoxia, PAD2 inhibited SERCA2a activity through citrullination. AAV9-mediated overexpression of PAD2 in cardiomyocytes worsened both survival rates and the incidence of ventricular arrhythmia following hemorrhagic shock in mice. Conversely, PAD2 enzymatic activity mutations and a PAD2-specific inhibitor improved survival rates and reduced arrhythmia after hemorrhagic shock. CONCLUSION: During myocardial hypoxia occurs in hemorrhagic shock, PAD2 reduces SERCA2a enzyme activity by citrullination, disrupting myocardial calcium homeostasis. Peptidylarginine deiminase 2 gene deficiency or inhibition improves ventricular arrhythmias and increases survival following hemorrhagic shock. LEVEL OF EVIDENCE: Original Research-basic sciences research; not applicable.