["The Journal of Physiology, Volume 604, Issue 7, Page 2683-2697, 1 April 2026. ", "\nAbstract figure legend The pharmacokinetics of lipophilic cationic radiotracers such as [99mTc]sestamibi and [99mTc]tetrofosmin are dependent on the electrical potentials that determine their distribution across the sarcolemmal and mitochondrial membranes. Fitting their temporal kinetics to a mathematical model that is reparametrized using the Nernst equation allows an estimation of these voltages ex vivo in the Langendorff perfused heart under baseline conditions, under hyperkalaemic depolarization and mitochondrial uncoupling with carbonyl cyanide 3‐chlorophenylhydrazone. Using planar scintigraphy, the method can be extended to give an independent estimation of these fundamental physiological parameters in vivo. Created in BioRender.\n\n\n\n\n\n\n\n\n\nAbstract\nTransmembrane electrical potentials across the sarcolemmal (Em${{E}_{\\mathrm{m}}}$) and mitochondrial (ΔΨm$\\Delta {{{{\\Psi}}}_{\\mathrm{m}}}$) membranes are central to cellular excitability, metabolism and viability. However, their direct and quantitative measurement in vivo remains challenging. We established a quantitative kinetic modelling framework to estimate Em${{E}_{\\mathrm{m}}}$ and ΔΨm$\\Delta {{{{\\Psi}}}_{\\mathrm{m}}}$ independently from dynamic radiotracer data in the heart using the Nernst equation applied to the kinetics of the lipophilic cationic tracers [99mTc]sestamibi and [99mTc]tetrofosmin. Parameters were estimated from high‐temporal‐resolution time–activity curves using non‐linear least squares and Markov chain Monte Carlo (MCMC) fitting. Experiments were performed in isolated Langendorff‐perfused rat hearts under baseline, hyperkalaemic depolarization and mitochondrial uncoupling with carbonylcyanide‐3‐chlorophenylhydrazone (CCCP) and in vivo using planar scintigraphy. In perfused hearts, baseline potentials were Em=−65±7mV${{E}_{\\mathrm{m}}}=-65\\pm 7\\, {\\mathrm{mV}} $ and ΔΨm=−109±9mV$\\Delta {{{{\\Psi}}}_{\\mathrm{m}}}= -109 \\pm 9\\, {\\mathrm{mV}}$ (mean ± SD, n = 4). Increasing [K+] caused dose‐dependent depolarization of Em${{E}_{\\mathrm{m}}}$ in agreement with Goldman–Hodgkin–Katz predictions, whereas ΔΨm$\\Delta {{{{\\Psi}}}_{\\mathrm{m}}}$ remained stable. CCCP selectively depolarized ΔΨm$\\Delta {{{{\\Psi}}}_{\\mathrm{m}}}$ to −66±8mV$-66 \\pm 8\\, {\\mathrm{mV}} $ (300 nm) and −6±2mV$-6 \\pm 2\\, {\\mathrm{mV}} $ (600 nm) with minimal effect on Em${{E}_{\\mathrm{m}}}$. In vivo, potentials were Em=−61±8mV${{E}_{\\mathrm{m}}}= -61 \\pm 8\\, {\\mathrm{mV}}$ and ΔΨm=−151±13mV$\\Delta {{{{\\Psi}}}_{\\mathrm{m}}}= -151 \\pm 13\\, {\\mathrm{mV}}$ (n = 4), consistent with physiological values. This modelling approach enables the first non‐invasive, independent quantitative estimation of sarcolemmal and mitochondrial membrane potentials in vivo. It overcomes limitations of optical probes and, with high‐sensitivity single‐photon emission computed tomography and positron emission tomography (PET) systems (including total body PET), offers new opportunities to assess bioenergetic dysfunction in cardiovascular disease and beyond.\n\n\n\n\n\n\n\n\n\nKey points\n\nPharmacokinetic modelling of [99mTc]sestamibi and [99mTc]tetrofosmin allowed independent estimation of sarcolemmal (Em${{E}_{\\mathrm{m}}}$) and mitochondrial (ΔΨm$\\Delta {{{{\\Psi}}}_{\\mathrm{m}}}$) membrane potentials ex vivo in the Langendorff perfused rat heart and in vivo in the rat heart.\nThe method gave independent measures of membrane potentials ex vivo when depolarized with hyperkalaemic buffers or mitochondrial uncoupling.\nIn vivo measurements of membrane potentials agreed with literature values, whereas ΔΨm$\\Delta {{{{\\Psi}}}_{\\mathrm{m}}}$ was found to be less polarized ex vivo in the perfused heart.\nThe method uses clinically available single‐photon emission computed tomography imaging agents that could be employed to measure these parameters in humans.\n\n\n"]