NETosis and Myeloperoxidase Promotes Inflammation and Cardiac Remodeling in Arrhythmogenic Cardiomyopathy

Abstract Background Arrhythmogenic cardiomyopathy (ACM) is a heritable nonischemic cardiomyopathy and a leading cause of sudden cardiac death. Although inflammation is a pathological hallmark of ACM, the contribution of peptidylarginine deiminase 4 (PAD4)-dependent neutrophil extracellular trap (NET) formation and myeloperoxidase (MPO) to disease progression remains poorly defined. Methods To define the role of PAD4-dependent NETosis and MPO signaling in ACM disease progression homozygous desmoglein-2 mutant ( Dsg2 mut/mut ) mice were utilized. We employed genetic and pharmacological approaches to determine the efficacy of targeting PAD4 and MPO on cardiac function, arrhythmogenic burden, myocardial fibrosis, inflammatory signaling, and gap junction integrity. Cardiac phenotyping included echocardiography, electrocardiography, histology, inflammatory profiling, and biochemical assays. Results Markers of PAD4-dependent NETosis were elevated in Dsg2 mut/mut hearts as early as 4 weeks of age, prior to cardiac dysfunction. Genetic deletion of Pad4 significantly preserved left ventricular function, reduced ectopics, attenuated myocardial fibrosis, and suppressed proinflammatory and profibrotic cytokines. MPO levels were increased in Dsg2 mut/mut hearts, and genetic ablation of Mpo preserved cardiac function, reduced arrhythmic burden, prevented myocardial fibrosis, and restored connexin-43 phosphorylation and localization. Furthermore, pharmacological MPO-inhibition improved cardiac function, reduced arrhythmias, and attenuated inflammatory signaling, though myocardial fibrosis was not fully prevented. Notably, hearts from patients with ACM demonstrated increased MPO signal in both cardiomyocytes and non-cardiomyocyte populations compared with donor controls. Conclusions PAD4-dependent NETosis and MPO signaling are key drivers of inflammation, fibrosis, and arrhythmogenesis in early disease onset in ACM. Targeting neutrophil-mediated pathways represents a promising therapeutic strategy to mitigate disease progression in ACM. Clinical Perspective What Is New? PAD4-dependent NET formation is activated early in ACM and directly contributes to myocardial inflammation, fibrosis, arrhythmias, and cardiac dysfunction. Genetic ablation of Pad4 or Mpo preserves cardiac function, reduces arrhythmogenic burden, and attenuates proinflammatory and profibrotic signaling in a Dsg2 mutant model of ACM. Pharmacological inhibition of MPO improves cardiac function and electrical stability, identifying neutrophil-derived pathways as modifiable drivers of disease. What Are the Clinical Implications? Neutrophil-mediated inflammation represents a clinically relevant mechanism in ACM that may be targeted without global immunosuppression. MPO inhibition may offer a novel disease-modifying strategy to reduce arrhythmias and preserve cardiac function in patients with ACM. Neutrophil- and NET-associated biomarkers may improve early risk stratification and therapeutic decision-making in genetically susceptible individuals. Graphical Abstract (A) Signaling pathway for PAD4-dependent NETosis. (B) Illustration of neutrophil undergoing NETosis resulting in the release of MPO and DNA histone complexes. (C) Effects of MPO release on cardiac tissue of ACM mice