Abstract Body (Do not enter title and authors here): Background
Fibrosis is a hallmark pathological feature of cardiac disease, yet FDA-approved drugs that specifically target cardiac fibrosis are lacking. Using preclinical mouse models, we discovered that genetic or pharmacological inhibition of Anthrax Toxin Receptor 1 (ANTXR1) alleviates heart failure following myocardial infarction (MI) or pressure overload. Here we set out to unravel the underlying mechanisms.
Methods
Single-cell RNA sequencing (scRNA-seq) was performed on murine hearts following angiotensin II/phenylephrine (ATII/PE)-induced hypertension or coronary artery ligation-induced MI, with or without ANTXR1 antibody (T8Ab) treatment. The impact of Col1α2-Cre–mediated Antxr1 conditional knockout (CKO) on cardiac function was also tested in these models. In vitro, Antxr1 wildtype and knockout (KO) immortalized cardiac fibroblasts (iCF) were established. The effects of ANTXR1 blockade under TGF-β1 stimulation were evaluated using collagen gel contraction, Western blot, and immunofluorescence (IF). ANTXR1–TGFBR1 interaction was assessed by co-IF, co-immunoprecipitation, and proximity ligation assay.
Results
scRNA-seq revealed high Antxr1 expression in cardiac fibroblasts (CF), with transcript levels peaking by day 14 post-MI. Antxr1⁺ fibroblasts increased ~9-fold by day 14 (17.8%) post-MI, and ~13-fold by day 28 post-ATII/PE (26.4%) versus controls (~2%). Co-IF confirmed ANTXR1 colocalization with PDGFRα, COL1, and THBS4 in hearts from both models. T8Ab treatment had no major impact on gene expression in healthy mice but significantly reduced expression of CF activation and ECM remodeling genes (e.g., Acta2, Meox1, Col1a1/2, Mmp14, Postn) post-MI. IF staining showed >50% reduction in denatured collagen, nuclear p-SMAD3, and YAP1 levels in CF from both models. Genetic disruption of Antxr1 in CF improved cardiac function following ATII/PE (EF%: WT 59.3% vs. CKO 75.9%) and MI (EF%: WT 46.4% vs. CKO 63.8%). In iCFs, ANTXR1 protein increased ~10-fold after serum starvation. ANTXR1 inhibition (KO or T8Ab) significantly suppressed iCFs activation as shown by reduced collagen gel contraction and levels of TGFBR1, p-SMAD2/3, and YAP1. ANTXR1 bound TGFBR1 to stabilize TGF-β signaling in mouse and human CF.
Conclusion
Blocking ANTXR1 alleviates heart failure in mice by preventing maladaptive TGFβ signaling in CF, highlighting ANTXR1 as a potential therapeutic target for cardiac fibrosis.