PROJECT SUMMARY Coronary artery disease (CAD) is the leading cause of death worldwide and poses a significant burden on healthcare and patient quality of life. It is caused by pathology of the coronary arteries that occludes blood flow to ventricular heart muscle. Existing revascularization treatments, such as coronary artery bypass grafts (CABG) or percutaneous coronary interventions, are not an option for all patients and have significant failure rates. One alternative approach could be induction of collateral arteries, which are a rare subset of arteries that bridge two conventional arteries and provide an alternative route for blood flow (i.e., natural bypasses). A small subset of human patients develops collateral arteries, and elevated collateral blood flow increases survival in humans. Thus, stimulating collateral arteries could be a useful treatment for CAD, yet their developmental mechanisms and biological functions are poorly understood. Our laboratory will utilize the normal biology of guinea pig hearts to discover mechanisms of collateral artery development. Guinea pigs are completely resistant to permanent coronary artery occlusion due to numerous collateral arteries, which are absent in most mammals including mice. In preliminary data, our laboratory discovered that guinea pig collateral arteries develop during embryonic development, a feature that will greatly facilitate discovery. We propose to use a comparative biology approach that uncovers collateral artery developmental mechanisms by comparing guinea pig and mouse embryonic hearts. (Aim1) We will use whole mount immunohistochemistry and single cell RNA sequencing to perform cellular resolution comparisons between developing guinea pig and mouse hearts. Genes and pathways correlating with collateral arteries (i.e. guinea pig hearts) will be considered candidate drivers of their development. (Aim2) We will next functionally test these candidates by introducing them into neonatal mouse hearts and assessing whether they induce collateral artery development. Upon completion of these aims, we will have fully established the guinea pig as a model to aid the study of collateral arteries. This includes compiling a guinea pig heart cell atlas, identifying how the guinea pig heart compares with mouse, and testing select candidate collateral inducers. Success will motivate a longer-term project with translational implications for CAD.