Abstract Body: Adverse fibrotic remodeling contributes to high morbidity and mortality in patients following myocardial infarction (MI). There is evidence that cardiac fibroblasts (CFs) play a role in every stage of healing post MI, which is due to their plastic nature allowing the cells to constantly adapt their phenotype and function to the rapid changes in the MI environment. While a myriad of stimuli driving fibrotic remodeling post-MI have been identified, the contribution of specific cell populations to the signaling cascade and how these communication networks are tuned in response to chronic stress remains unclear. The cytoskeletal protein, β_IV-spectrin, coordinates a signaling complex with the transcription factor, STAT3, to modulate CF activation and profibrotic signaling. Specifically, stress-induced loss of β_IV-spectrin promotes subcellular redistribution and activation of STAT3. Mice expressing degradation-resistant β_IV-spectrin (qv^3J) show increased mortality and incidence of cardiac rupture within the first week after permanent occlusion of the left anterior descending artery. Histology at 7 days post-MI shows less fibrosis in the qv^3J infarct region compared to WT. Therefore, we hypothesized that stress-induced loss of β_IV-spectrin is an important step in CFs activation and scar formation. We subjected WT and qv^3J mice to MI and assessed cardiac function and survival through 28 days and performed spatial transcriptomics at 7 days. Spatial transcriptomics analysis revealed two subpopulations of CFs that showed differential expression of the collagen triple helix repeat containing-1 (Cthrc1^low and Cthrc1^high). A STAT3-dependent fibroblast trajectory was identified that originates from vascular smooth muscle cells and progresses to Cthrc1^high CF population in WT hearts, while the qv^3J fibroblast trajectory diverged into a sustained proliferative state. The infarct and border zone showed high expression of STAT3 related genes, fibrotic genes, and MMPs in WT hearts. Despite qv^3J hearts having decreased bulk expression of STAT3 and MMP, spatial transcriptomics revealed enrichment of MMP expression in the border zone region, resulting in a steep spatial gradient of MMP activation. Zymography confirmed increased MMP activity in isolated qv^3J CFs from infarcted hearts. Our findings reveal a novel molecular pathway critical for proper spatiotemporal control of the healing process following MI that could be targeted for future therapies.