AbstractBy the time cardiotoxicity-associated cardiac dysfunction is detectable by echocardiography it is often beyond meaningful intervention. ^99mTc-sestamibi is used clinically to image cardiac perfusion by single photon emission computed tomography (SPECT) imaging, but as a lipophilic cation its distribution is also governed by mitochondrial membrane potential (ΔΨ_m). Correcting scans for variations in perfusion (using a ΔΨ_m-independent perfusion tracer such as (bis(N-ethoxy-N-ethyldithiocarbamato)nitrido ^99mTc(V)) (^99mTc-NOET) could allow ^99mTc-sestamibi to be repurposed to specifically report on ΔΨ_m as a readout of evolving cardiotoxicity. Isolated rat hearts were perfused within a γ-detection apparatus to characterize the pharmacokinetics of ^99mTc-sestamibi and ^99mTc-NOET in response to mitochondrial perturbation by hypoxia, ionophore (CCCP) or doxorubicin. All interventions induced ^99mTc-sestamibi washout; hypoxia from 24.9 ± 2.6% ID to 0.4 ± 6.2%, CCCP from 22.8 ± 2.5% ID to −3.5 ± 3.1%, and doxorubicin from 23.0 ± 2.2% ID to 17.8 ± 0.7, p < 0.05. Cardiac ^99mTc-NOET retention (34.0 ± 8.0% ID) was unaffected in all cases. Translating to an in vivo rat model, 2 weeks after bolus doxorubicin injection, there was a dose-dependent loss of cardiac ^99mTc-sestamibi retention (from 2.3 ± 0.3 to 0.9 ± 0.2 ID/g with 10 mg/kg (p < 0.05)), while ^99mTc-NOET retention (0.93 ± 0.16 ID/g) was unaffected. ^99mTc-NOET therefore traps in myocardium independently of the mitochondrial perturbations that induce ^99mTc-sestamibi washout, demonstrating proof-of-concept for an imaging approach to detect evolving cardiotoxicity.