Peripheral arterial disease (PAD) is caused by atherosclerosis, the buildup of plaque that can obstruct blood flow in the arteries to the lower extremities. The current assessment of patients with PAD targets the anatomic or hemodynamic burden of atherosclerotic plaque stenosis with measurement of ankle-brachial index (ABI), and several imaging other techniques. However, anatomic and hemodynamic indices do not always correlate with the functional limitations and disability that PAD patients experience, and prior work suggests that the PAD population would benefit from more specific functional tissue tests. We hypothesize that metabolic maps of phosphocreatine (PCr) measures, reflecting severe skeletal muscle (SM) ischemia or downstream mitochondrial changes, may fill that gap. PCr is the most abundant high- energy phosphate present in muscle. Energy metabolism and PCr play a vital role in cellular homeostasis, but there currently are no routine diagnostic tests to noninvasively quantify or map the distribution of PCr in patients with PAD. Phosphorus (31P) magnetic resonance spectroscopy (MRS) is arguably the gold standard for the noninvasive assessment of SM mitochondrial function and high-energy phosphate content. However, due to the relatively low MR detection sensitivity and the requirement for unique hardware, 31P MRS is not used in routine clinical applications. Chemical exchange saturation transfer (CEST) MRI has emerged as a novel, high-sensitivity technique that may overcome several of the limitations of 31P MRS. However, CEST MRI is still under development and one major impediment for more widespread application is limited specificity for a particular metabolite due to spectral overlap of CEST signal from other metabolites and proteins and as well as the background signal from semi-solid macromolecules and direct saturation of water Our long-term goal is to develop clinically translatable CEST methods to extract and quantity PCr concentrations in skeletal muscle that provides a sensitive MRI approach to assess SM metabolism. If successful, this new technique should provide a completely new and sensitive method for detecting PCr in calf muscle and may play a pivotal role for the evaluation of regional musle pathophysiology change in many musculoskeletal diseases. We recently developed two new CEST techniques, dubbed as polynomial and Lorentzian line-shape fitting (PLOF) method and artificial neural network based CEST quantification method (ANNCEST) that are able to detect PCr signal with high sensitivity and specificity. We will develop and optimize the PLOF and ANNCEST methods for PCr mapping through one novel animal model and in-magnet plantar flexion exercise for human leg. The optimized CEST MRI methods will be applied on PAD patients to validate that PCr dynamic curve is correlated with the severity of the PAD. Upon the successful completion of this proposal, we anticipate developing the first rapid, high-resolution skeletal muscle...