This proposal?s objective is to develop and evaluate the microSPECT measurement of myocardial blood flow (MBF) in mice. MBF is a measure of microvasculature health and is an independent predictor of cardiovascular morbidity and mortality. Microvasculature dysfunction is often called coronary microvascular disease (CMVD) and has become an increasingly well-recognized cardiac pathology. Yet, despite mounting clinical evidence of its importance, there is a dearth of mechanistic understanding and targeted therapies for CMVD. The availability of numerous disease-based and genetic models and the ease of genetic manipulations makes the mouse model ideal for studying cardiovascular disease and its treatment. To develop non-invasive MBF measures in mice, we plan to consider 99mTc-labeled sestamibi and 201Tl as tracers; each has its own flow-dependent tracer extraction fraction (EXF), which can be species-specific. Thus, in Aim 1, we will map the relationship between radiotracer uptake, as measured by K1, and MBF for both tracers in normal mice. We will validate the use of microspheres as our gold-standard measure for MBF in mice by measuring the uncertainty of injections into the left-ventricle (LV) and left atrium (LA), where the latter is desirable for the more uniform mixing with blood before the microspheres are ejected from the LV, but is more technically challenging due to the small size of the LA and its thin wall. Similarly, we will use a dose calibrator to measure the tracer?s uptake concentration in the excised heart. With these gold standards for comparison, we will develop microSPECT MBF quantification on the MI Labs U-SPECT+ using a unique phantom and targeted animal studies to develop quantitative corrections and optimizations (Aim 2). This system can achieve resolution of ~0.35 mm with high sensitivity. The phantom will be used for studying the crosstalk relationship between uptake in the myocardium and the input function, as measured in the LV. We will optimize our dynamic fitting procedures using this phantom and determine appropriate quantitative corrections that account for spillover due to the system?s spatial resolution. This will be tested in our evaluations (Aim 3) where we will compare the longitudinal myocardial blood flow reserve (MBFR) in wild-type mice and transgenic mice in which the degree of microvascular dysfunction can be regulated. These longitudinal studies will be complemented with microsphere MBF and histological measures of capillary density. We expect the project?s outcomes to be: (1) microsphere MBF validation in mice and uncertainty determination; (2) EXF curves for sestamibi and 201Tl in normal mice; (3) determination of the better tracer for MBFR measurements in mice; (4) validation of that tracer?s EXF curve in a transgenic model; (5) development and validation of accurate in vivo MBF/MBFR measurements in mice; (6) validation of imaging-based MBF/MBFR decline in transgenic mice with gene activation; and (7) correlation of imaging and microsphere MBF/MBFR with capillary density. Developing this imaging technique in mice combined with a unique disease model that can be regulated will provide a powerful tool for research into coronary microvasculature disease.