Angiogenesis is a vital component of the wound healing response after myocardial infarction (MI), mitigating scarring and worsening of heart function. Endothelial cell (EC) functional heterogeneity after MI is incompletely understood. We subjected wild-type (WT) mice to transient coronary artery ligation or sham surgery and performed single cell RNA sequencing on ECs isolated from the left ventricle on day 3. Unsupervised clustering identified 12 distinct subpopulations. One cluster was highly proliferative (GO-term analysis using PantherDB) and strongly expanded after MI (9.2-fold increase vs. sham). Prnd (prion-like protein doppel) was one of the most highly enriched genes in this population. Prnd encodes a 20 kDa protein (PRND) attached to the outer leaflet of the cell membrane via a GPI-anchor. PRND displays structural similarities to the prion protein but is unable to misfold into an infectious, prion disease-promoting protein conformation. PRND was weakly expressed in the heart under sham-operated baseline conditions but strongly upregulated in the infarct region after MI (14.9-fold vs. sham, peak on day 3, P<0.001). Immunofluorescence microscopy indicated that PRND was expressed in a subpopulation of ECs in the infarct region but not in ECs in the remote region. Consistent with low basal PRND expression, Prnd deficient (global knockout, KO) mice displayed no apparent cardiac phenotype under baseline conditions. After MI, however, Prnd KO mice developed larger scars than their WT littermates (36 ± 3 vs. 20 ± 3% of the left ventricle on day 28; n=8–10; P=0.005) with more pronounced systolic dysfunction (echocardiographic left ventricular fractional area change on day 28, 16 ± 2 vs. 25 ± 2; n=8–10; P=0.034). Adverse post-MI remodeling in Prnd KO mice was associated with a reduced capillary density in the infarct border zone which became apparent on day 3 (ECs per cardiomyocyte KO vs. WT, 1.07 ± 0.04 vs. 1.20 ± 0.02; n=3; P=0.035) and was sustained on day 28 (1.18 ± 0.03 vs. 1.37 ± 0.01; n=6; P<0.001). In the cancer field, PRND is known to facilitate receptor tyrosine kinase (RTK)-mediated signal transduction. We identified the stem cell factor (SCF) and meteorin-like (METRNL) receptor Kit as the most highly enriched RTK in Prnd-expressing ECs after MI. Cardiac ECs isolated from Prnd KO mice displayed a reduced proliferative capacity in response to SCF and METRNL compared with ECs isolated from WT littermates. Conversely, lentiviral overexpression of PRND in human coronary artery endothelial cells augmented cell proliferation in response to SCF and METRNL. In HEK-293 cells expressing PRND and KIT, immunoprecipitation using a monoclonal KIT antibody co-purified PRND, suggesting a physical interaction between GPI-anchored PRND and the KIT receptor. In conclusion, the cell membrane-attached protein PRND is preferentially expressed by proliferating ECs, promotes angiogenesis and functional adaptation after MI, and facilitates angiogenic signaling via KIT.
