**Background:** Heart failure (HF) affects approximately 26 million people worldwide, and myocardial inflammation—particularly sterile inflammation mediated by mitochondrial DNA (mtDNA) acting as a damage-associated molecular pattern—plays a critical role in its pathophysiology. mtDNA activates pattern recognition receptors such as TLR9, leading to NF-κB and NLRP3 inflammasome activation and increased inflammatory cytokines including IL-1β and IL-6. ZBP1 (Z-DNA binding protein 1) is a nucleic acid sensor known to positively regulate inflammation in response to mtDNA in inflammatory cells, fibroblasts, and endothelial cells. However, its role in cardiomyocytes and the heart was unknown. This study investigated the role of ZBP1 in mtDNA-induced myocardial inflammation and HF development using both in vitro and in vivo approaches.
**Methods:** mtDNA (1000 ng/mL) extracted from rat liver or heart was administered to cultured neonatal rat ventricular myocytes (NRVMs) and RAW264.7 macrophages. ZBP1 was knocked down using siRNA or overexpressed using adenovirus. RIPK3, NF-κB p65, TLR9, RIPK1, and STING were also knocked down using siRNA. CpG-oligodeoxynucleotides (CpG-ODN) and control-ODN were used to stimulate TLR9. Myocardial infarction (MI) was induced by left anterior descending artery ligation in wild-type (WT) C57B/6J mice and ZBP1 knockout (KO) mice. Pressure overload was induced by transverse aortic constriction. Protein and mRNA levels were measured by immunoblotting and real-time PCR. Cardiac function was assessed by echocardiography at 3 and 28 days post-MI. Histological analysis included Masson-trichrome staining for fibrosis, TUNEL staining for apoptosis, and immunohistochemistry for ZBP1 and RIPK3.
**Key Results:** In cardiomyocytes, mtDNA (1000 ng/mL) increased ZBP1 mRNA and protein levels, along with RIPK3, phosphorylated NF-κB (Ser536), NLRP3, phosphorylated TBK1, IL-1β, and IL-6. A low dose (100 ng/mL) increased LC3-II but not ZBP1 or inflammatory cytokines. Unexpectedly, ZBP1 knockdown exacerbated mtDNA-induced increases in RIPK3, phosphorylated NF-κB, NLRP3, IL-1β, and IL-6, while ZBP1 overexpression attenuated them. In contrast, ZBP1 knockdown in macrophages attenuated mtDNA-induced IL-1β and IL-6, confirming a proinflammatory role in macrophages. RIPK3 knockdown canceled the exacerbation of phosphorylated NF-κB, NLRP3, IL-1β, and IL-6 by ZBP1 knockdown in mtDNA-treated cardiomyocytes. NF-κB knockdown suppressed NLRP3, IL-1β, and IL-6 increases by ZBP1 knockdown. ZBP1 knockdown did not affect TBK1 phosphorylation or cell viability. CpG-ODN increased RIPK3, IL-1β, and IL-6, and ZBP1 knockdown exacerbated these responses. TLR9 knockdown enhanced mtDNA-induced ZBP1 increases and attenuated RIPK3, phosphorylated NF-κB, NLRP3, IL-1β, and IL-6. ZBP1 colocalized with FITC-labeled CpG-ODN, and ZBP1 knockdown increased TLR9-CpG-ODN colocalization. In vivo, Dloop (mtDNA component) was increased in the cytosolic fraction of noninfarcted regions of post-MI mouse hearts compared with controls, while Tert and B2m (nuclear DNA components) were not. ZBP1, RIPK3, phosphorylated NF-κB, NLRP3, IL-1β, and IL-6 were increased in failing hearts. ZBP1 KO mice exhibited exacerbated LV dilatation (increased LVDd and LVDs), decreased LV ejection fraction and fractional shortening, increased heart weight and LV weight, increased interstitial fibrosis (collagen volume), and increased myocardial apoptosis (TUNEL-positive cells and BAX/Bcl2 ratio) compared with WT mice at 28 days post-MI. At 3 days post-MI, no significant differences in cardiac function or infarct size were observed between WT and ZBP1 KO mice. TBK1 phosphorylation was not significantly affected by ZBP1 KO.
**Clinical Implications:** This study reveals a novel, cell-type-specific role for ZBP1 as an endogenous suppressor of mtDNA-induced myocardial inflammation in cardiomyocytes, acting through inhibition of the RIPK3-NF-κB-NLRP3 pathway, which is opposite to its proinflammatory role in macrophages. The finding that ZBP1 sequesters cytosolic mtDNA and prevents TLR9 activation suggests a protective mechanism against sterile inflammation in the heart. ZBP1 and cytosolic mtDNA are increased in failing hearts, and ZBP1 deficiency exacerbates cardiac remodeling and dysfunction after MI. These results identify ZBP1 as a potential therapeutic target for preventing cardiac remodeling and heart failure by modulating myocardial inflammation. However, the cell-type-specific effects of ZBP1 (anti-inflammatory in cardiomyocytes vs. proinflammatory in macrophages) must be carefully considered in any therapeutic strategy.