'Youthful' phenotype of c-Kit⁺ cardiac fibroblasts

Cardiac fibroblast (CF) population heterogeneity and plasticity present a challenge for categorization of biological and functional properties. Distinct molecular markers and associated signaling pathways provide valuable insight for CF biology and interventional strategies to influence injury response and aging-associated remodeling. Receptor tyrosine kinase c-Kit mediates cell survival, proliferation, migration, and is activated by pathological injury. However, the biological significance of c-Kit within CF population has not been addressed. An inducible reporter mouse detects c-Kit promoter activation with Enhanced Green Fluorescent Protein (EGFP) expression in cardiac cells. Coincidence of EGFP and c-Kit with the DDR2 fibroblast marker was confirmed using flow cytometry and immunohistochemistry. Subsequently, CFs expressing DDR2 with or without c-Kit was isolated and characterized. A subset of DDR2⁺ CFs also express c-Kit with coincidence in ~ 8% of total cardiac interstitial cells (CICs). Aging is associated with decreased number of c-Kit expressing DDR2⁺ CFs, whereas pathological injury induces c-Kit and DDR2 as well as the frequency of coincident expression in CICs. scRNA-Seq profiling reveals the transcriptome of c-Kit expressing CFs as cells with transitional phenotype. Cultured cardiac DDR2⁺ fibroblasts that are c-Kit⁺ exhibit morphological and functional characteristics consistent with youthful phenotypes compared to c-Kit^- cells. Mechanistically, c-Kit expression correlates with signaling implicated in proliferation and cell migration, including phospho-ERK and pro-caspase 3. The phenotype of c-kit⁺ on DDR2⁺ CFs correlates with multiple characteristics of 'youthful' cells. To our knowledge, this represents the first evaluation of c-Kit biology within DDR2⁺ CF population and provides a fundamental basis for future studies to influence myocardial biology, response to pathological injury and physiological aging.