Despite decades of advances in clinical diagnosis and treatment of heart disease, there is a rising epidemic of heart failure in an aging population worldwide. Historically, much focus has been on atherosclerosis, acute myocardial infarction, and treatment of large vessel coronary disease. However, relatively little is known at a mechanistic level about the vast network of small arteries, capillaries and veins in the heart beyond the large coronary arteries, the coronary microcirculation, and increasing evidence links dysfunction of the microcirculation to various forms of heart disease including heart failure. In vitro experiments, histologic analysis, and computational modeling have provided insight about how the coronary microcirculation is regulated and remodels in the failing heart, but prior studies have been unable to directly evaluate the complex microcirculatory physiology of the heart at the cellular level in vivo. Intravital optical microscopy is being used in the neurosciences and tumor biology to decipher dynamic vascular physiology in vivo, but these techniques have not been applicable in the heart due to severe imaging limitations imposed by contractile motion. We have recently pioneered intravital imaging methods to perform motion-artifact free, cellular resolution microscopy in the beating heart. Building on this work, this proposal seeks to investigate mechanisms of coronary microvascular dysfunction by utilizing intravital microscopy to quantitatively map flow in the coronary microcirculation down to the capillary level in animal models of heart disease. Studies will be performed in mice comparing microcirculatory function in healthy controls, in a model of physiologic hypertrophy due to exercise, and in a model of pressure overload leading to pathologic hypertrophy and heart failure. In addition, the specific role of microvascular pericytes will be investigated as a master regulator of capillary blood flow. Intravital confocal and two-photon microscopy, multiplexed fluorescent reporters, and cell-specific optogenetics approaches will be used to record and manipulate microvascular flow, and to correlate abnormal flow patterns with cardiomyocyte metabolism, inflammatory response, and fibrosis. This work will lead to new understanding of microcirculatory pathophysiology in the failing heart at the single cell level and promising new insights for clinical therapeutics.