Small Peptide Antagonists Derived Based on in Silico Analysis Block CXCL10‐CXCR3 Signaling and Function on Cardiac Fibroblasts and Cardiomyocytes

Myocarditis is an inflammatory disease of the heart muscle caused by a viral infection, which often results in cardiac dysfunction and death—especially in young patients. The severe cardiac inflammation that characterizes the pathogenesis of myocarditis is mediated by leukocytes entering the cardiac tissue, and is often accompanied by remodeling and cardiomyocyte apoptosis. Possible cell sources for viral replication are cardiac fibroblasts (CF) and cardiomyocytes (CM), thought to be important contributors of virus replication, which aggravate myocarditis. It has been recently observed that CXC chemokine ligand 10 (CXCL10) is unregulated in the myocardium during the early stages of infection. CXCL10 is thought to be the master regulator of myocardial interactions between cardiac and immune cell migration, perhaps affecting cardiac damage and the clinical progression of myocarditis. We have previously shown that CXCL10, via its receptor CXCR3, inhibits growth factor‐induced motility in both CF and CM. Currently, clinical therapies for myocarditis are predominantly based on symptoms and diagnosis of the underlying infection, but lack a targeted approach to inhibiting the cardiac damage induced by infections. As a first step to this approach, we designed, developed, and tested in vitro peptides with the potential to inhibit the cardiac damage induced by infections. In the present study we test two fifth‐generation small peptide antagonists of CXCL10‐CXCR3, 14 aa CR3‐5‐3 and CR3‐5‐5, developed by in silico prediction‐based functional peptide design to directly bind to CXCL10 ( Figure 1). We hypothesize that CR3‐5‐3 and CR3‐5‐5 peptides will similarly block CXCL10‐CXCR3 signaling and function on cardiac fibroblasts and cardiomyocytes. CM and CF were isolated from wild type and CXCR3‐null FVB neonatal mice. Following isolation, CM, CF, or CM/CF in both direct and indirect co‐cultures were challenged with CM and CF activating growth factor TGF‐β. Antagonist peptide treatments, CR3‐5‐3 and CR3‐5‐5, were given alone or in combination with CXCL10. To determine whether CR3‐5‐3 and CR3‐5‐5 blocks CXCL10 binding to its receptor, we analyzed the ability of biotin‐tagged CXCL10 and/or FITC‐tagged CR3‐5‐5 to compete for binding to CM and CF using flow cytometry. In vitro cellular function assays were assessed for migration, apoptosis, and ECM secretion. Real time quantitative polymerase chain reaction (RT‐PCR) was employed in order to determine modified relative gene expression of both CM and FB, following treatment. Interestingly, CR3‐5‐3 and CR3‐5‐5 alter CXCL10‐induced gene expression on both FB and CM. We found that CR3‐5‐5 interferes with CXCL10 inhibition of TGF‐β‐induced matrix production. Meanwhile, CR3‐5‐3 and CR3‐5‐5 altered CXCL10 inhibition of TGF‐β‐induced P38 Mitogen‐Activated Protein Kinase (MAPK)‐dependent extracellular matrix production in cardiac fibroblasts. Histochemical and fluorescence analyses of CM/CF co‐cultures revealed ECM CR3‐5‐3‐mediated alterations following TGF‐β and/or CXCL10 treatment. We have concluded that our data shows CR3‐5‐3 and CR3‐5‐5 results in the disinhibition of CXCL10‐suppressed growth factor‐induced intracellular P38 signaling, motility, and ECM production. We demonstrate the utility of in silico peptide development to be effective for developing chemokine antagonists. The therapeutic benefits of CR3‐5‐3 and CR3‐5‐5 on CXCL10, a master regulator of myocardial interactions between cardiac and immune cells in the progression of myocarditis, seems plausible and will be explored in future studies. Support or Funding Information American Society for Investigative Pathology‐Summer Research Opportunity in Pathology Program (SROPP) and University of Pittsburgh School of Nursing.Binding of CR3‐5‐5 to CXCL10