Myocardial infarction (MI) represents the most prevalent and severe manifestation of coronary artery disease worldwide. Following MI, the heart initiates a pro-inflammatory response to clear necrotic tissue, followed by an anti-inflammatory phase to promote the subsequent healing process by collagen deposition and scar formation. However, imbalanced or excessive inflammatory responses can exacerbate infarct size and impair heart function. As the first immune cells to infiltrate the infarcted heart, neutrophils play dual roles in both promoting inflammation and facilitating its resolution, including upregulating extracellular matrix protein (ECM) genes that can contribute to cardiac fibrosis and adverse cardiac remodelling. In a previous study we found ADAM10 to regulate the CX3CL1/ CX3CR1 chemokine axis and promote neutrophil recruitment in the infarcted heart. In this study, we investigated the contribution of neutrophils to extracellular matrix (ECM) production during post-infarction scar formation and analysed neutrophils ECM gene expression profiles in wild-type and MI mice using multi-omics, spectral flow cytometry, immunofluorescence, immunohistochemistry, and decellularized tissue staining approaches.
Transcriptomics and proteomic datasets of isolated neutrophils and whole-heart tissues from infarcted mouse models were analysed to identify upregulated ECM genes at various time points post-MI. Multi-omics profiling revealed that neutrophils upregulated Col1a1, Col3a1, Emilin1, Postn, Spp1, Fn1, and Agrn as early as day 1, and increased significantly at day 3, and 7 after infarction. The same genes were also upregulated in the whole-heart tissues at day 3 after MI. Interestingly, the ECM gene expression was significantly downregulated in infarcted mice treated with ADAM10 inhibitor. Mass spectrometry results further confirmed that ADAM10 inhibition significantly reduced ECM protein deposition at day 28 after MI. Flow cytometry analysis showed that cardiomyocyte-specific ADAM10-knockout significantly reduced infiltration of CX3CR1+ neutrophils 3 days after MI. Separate flow cytometry revealed that the CX3CR1+ neutrophils also co-express a pro-inflammatory and pro-fibrotic marker SiglecF, accumulating by day 3 and persisted up to 28 days after infarction in the heart. Further validation using RT-qPCR and immunofluorescence staining to compare the gene and protein expression profile in wild-type and MI mice demonstrated that Col1a1, Postn, Fn1, and Agrn, were more likely to be induced by neutrophil N1 pro-inflammatory phenotype in response to injury.
Taken together, these findings underscore neutrophils as active contributors to ECM remodelling after MI through upregulation of ECM-related genes in response to injury. Targeting CX3CR1+ neutrophils by inhibition of ADAM10 prevents the shedding of the chemokine CX3CL1, thereby limiting excessive inflammation and promoting a more balanced scar formation after infarction.
