Rationale: The subtle hypoxia underlying chronic cardiovascular disease is an attractive target for PET imaging, but the lead hypoxia imaging agent 64CuATSM, originally intended for cancer imaging, detects degrees of hypoxia far more extreme than would occur in the heart clinically. We are therefore developing analogs of 64CuATSM better suited to identify compromised but salvageable myocardium at the point where it becomes vulnerable, identified and validated using parallel biomarkers of cardiac function, energetics and biochemistry comparable to those observed in cardiac patients. Methods and Results: Isolated rat hearts (male Wistar 250g, n=6/group) were perfused with aerobic buffer for 20 min, then a range of hypoxic buffers (using a computer-controlled gas mixer) for 45 min. Contractility was monitored by intraventricular balloon, energetics by 31P NMR spectroscopy, lactate and creatine kinase release spectrophotometrically, and HIF1α by Western blotting of tissue homogenates. We identified a key hypoxia threshold at 30% buffer O2 saturation which induced a stable and potentially survivable functional and energetic compromise: LV developed pressure was depressed by 20%, and cardiac phosphocreatine was depleted by 65.5 ± 14% (mean +/- SD, p<0.05 vs control, ANOVA & t-test), but ATP levels were maintained. Lactate release was elevated (0.21 ± 0.067 versus 0.056 ± 0.01 mmol/L/min, p<0.05), but not maximal (0.46 ± 0.117 mmol/L/min at 0% buffer oxygen saturation), indicating residual oxidative metabolic capacity. HIF1α was also elevated, but not maximal. At this key threshold, 64CuCTS deposited significantly more 64Cu in the heart than any other tracer we examined, and more than twice that of 64CuATSM (61.8 ± 9.6% injected dose versus 29.4 ± 9.5% p<0.05). Conclusion: At the key hypoxia threshold required for maintained cardiac energetics, representative of survivable low grade cardiac ischaemia, only 64CuCTS delivered a hypoxic:normoxic contrast of 3:1, a prerequisite for successful PET imaging. We are now translating this approach to in vivo tracer evaluation with a view to progressing successful candidates such as 64CuCTS to clinical trial for imaging chronic cardiac ischemic syndromes.