Subacute Inhibition of the CD40 Pathway with TRAF-STOP Mitigates Fibrosis and Immune Cell Infiltration in Post-Myocardial Infarction Cardiac Remodeling

Background: The tissue response following acute myocardial infarction (AMI) entails distinct phases, each involving infiltration and activity of different sets of immune cells. The initial acute phase is marked by a pro-inflammatory response, which is followed by a resolution and repair phase in the infarcted area. Exacerbated and persistent inflammation during these phases is associated with adverse outcomes, including adverse cardiac remodeling, fibrosis, and ventricular dysfunction. The CD40-CD40L interaction regulates inflammatory responses via activation of the tumor necrosis factor (TNF) receptor-associated factors (TRAFs). TRAF-STOP, a small molecule we developed, inhibits the CD40-TRAF6 interaction, targeting the CD40-driven, pro-inflammatory pathway in monocytes and macrophages. This study aimed to explore the mechanisms behind CD40-TRAF6 inhibition, focusing on fibrosis and cardiac immune cell infiltration.

Methods: Male C57BL/6J mice (10-12 weeks) were subjected to temporary ligation (45 min) of the left anterior descending coronary artery to induce reperfused AMI. Starting on day 5 post-AMI, mice were treated with either TRAF-STOP or a control solution three times a week. Markers of fibrosis, scar size, and cardiac immune cells were assessed at 14 and 28 days using histological analysis and quantitative polymerase chain reaction (qPCR).

Results: Subacute inhibition of CD40 signaling with TRAF-STOP significantly reduced scar size (WGA signal per left ventricle (LV)) on day 14 (Control: 13.2 ± 3.5% vs. TRAF-STOP: 8.1 ± 0.9%, p = 0.0305) and day 28 (Control: 25.6 ± 1.8% vs. TRAF-STOP: 13.5 ± 2.5%, p = 0.0002). Fibrotic markers such as periostin (Control: 29.6 ± 5.2% vs. TRAF-STOP: 16.4 ± 5.4%, p = 0.0124) and fibroblast activation protein (FAP; Control: 25.5 ± 4.7% vs. TRAF-STOP: 9.7 ± 4.5%, p = 0.0029) were reduced as measured by qPCR and histology. Cardiac infiltration of CD45+ (-46.0%, p = 0.0490), CD19+ (-50.1%, p = 0.0194), CD4+ (-43.9%, p = 0.0491), and CD8+ (-34.6%, p = 0.0481) cells decreased at 14 days but not at 28 days as assessed by histology. Additionally, CD31+ cells, a marker of angiogenesis, were reduced after 14 days (-72.6%, p = 0.0002) but not at 28 days.

Conclusion: Subacute and continuous inhibition of the CD40 pathway reduces immune cell infiltration and limits the pro-fibrotic response following AMI. These findings identify the mechanism of TRAF-STOP-mediated restoration of left ventricular function post-AMI, potentially offering a new therapeutic strategy for managing post-AMI cardiac remodeling and preservation of cardiac function.