Neutrophil CAPNS1 regulates cardiac inflammation and injury by promoting NETosis in CVB3-induced myocarditis.

Exploring the immune mechanisms underlying Coxsackievirus B3 (CVB3)-induced myocarditis is critical for developing effective therapies, and the calpain small subunit 1 (CAPNS1) has emerged as a key regulator of inflammatory and immune responses. We investigated the regulatory role of neutrophil CAPNS1 in neutrophil recruitment, inflammatory cytokine production, and neutrophil extracellular trap (NET) formation. We observed that CVB3 infection significantly enhanced neutrophil recruitment to the heart and systemic NETs release in vivo, and induced NETosis in isolated bone marrow-derived polymorphonuclear neutrophils (BM-PMNs) in vitro. Pharmacological inhibition of NETs formation with Cl-amidine, a PAD4 inhibitor, markedly alleviated myocardial inflammation, injury, and fibrosis, reduced levels of cardiac injury markers and inflammatory cytokines, and preserved cardiac function in CVB3-infected mice. By profiling a single-cell sequencing dataset of viral myocarditis and subsequent validating, we found that CAPNS1 was the most significantly upregulated subtype of the calpain family in neutrophils, and was upregulated in cardiac-infiltrating neutrophils, from CVB3-infected mice and in CVB3-stimulated neutrophils in vitro. Conditional knockout of CAPNS1 in neutrophils using CAPNS1flox/flox; Lyz2-Cre mice ameliorated cardiac inflammation, fibrosis, and injury and improved cardiac function after CVB3 infection. To study the impact of neutrophils on other cardiac resident cells, conditioned medium from CAPNS1-knockout or DNase I-treated neutrophils inhibited cardiac fibroblast activation and cardiomyocyte apoptosis. We further demonstrated that CVB3 infection promoted Nesprin 1 downregulation and NETs formation, while CAPNS1 knockout or Nesprin 1 overexpression partially rescued the effects. Calpain activity was elevated in CVB3-infected hearts and neutrophils, which was partially reduced by myeloid-specific CAPNS1 deletion. Co-immunoprecipitation confirmed a direct interaction between Calpain 1 and Nesprin 1. Upregulation of CAPNS1 also showed increased degradation of Nesprin 1 after CAPN1 overexpression in BM-PMNs. Overall, these results establish that neutrophil CAPNS1 exacerbates CVB3-induced myocardial injury by promoting inflammatory cytokine release and NETs formation via calpain-mediated Nesprin 1 degradation. Targeting neutrophil CAPNS1 represents a promising novel therapeutic approach for viral myocarditis.