Sarcolemmal and mitochondrial membrane potentials measured ex vivo and in vivo in the heart by pharmacokinetic modelling of [99mTc]sestamibi.

Transmembrane electrical potentials across the sarcolemmal ( E m ${{E}_{\mathrm{m}}}$ ) and mitochondrial ( Δ Ψ m $\Delta {{{{\Psi}}}_{\mathrm{m}}}$ ) 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 E m ${{E}_{\mathrm{m}}}$ and Δ Ψ m $\Delta {{{{\Psi}}}_{\mathrm{m}}}$ 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 E m = - 65 ± 7 mV ${{E}_{\mathrm{m}}}=-65\pm 7\, {\mathrm{mV}} $ and Δ Ψ m = - 109 ± 9 mV $\Delta {{{{\Psi}}}_{\mathrm{m}}}= -109 \pm 9\, {\mathrm{mV}}$ (mean ± SD, n = 4). Increasing [K+] caused dose-dependent depolarization of E m ${{E}_{\mathrm{m}}}$ in agreement with Goldman-Hodgkin-Katz predictions, whereas Δ Ψ m $\Delta {{{{\Psi}}}_{\mathrm{m}}}$ remained stable. CCCP selectively depolarized Δ Ψ m $\Delta {{{{\Psi}}}_{\mathrm{m}}}$ to - 66 ± 8 mV $-66 \pm 8\, {\mathrm{mV}} $ (300 nm) and - 6 ± 2 mV $-6 \pm 2\, {\mathrm{mV}} $ (600 nm) with minimal effect on E m ${{E}_{\mathrm{m}}}$ . In vivo, potentials were E m = - 61 ± 8 mV ${{E}_{\mathrm{m}}}= -61 \pm 8\, {\mathrm{mV}}$ and Δ Ψ m = - 151 ± 13 mV $\Delta {{{{\Psi}}}_{\mathrm{m}}}= -151 \pm 13\, {\mathrm{mV}}$ (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. KEY POINTS: Pharmacokinetic modelling of [99mTc]sestamibi and [99mTc]tetrofosmin allowed independent estimation of sarcolemmal ( E m ${{E}_{\mathrm{m}}}$ ) and mitochondrial ( Δ Ψ m $\Delta {{{{\Psi}}}_{\mathrm{m}}}$ ) membrane potentials ex vivo in the Langendorff perfused rat heart and in vivo in the rat heart. The method gave independent measures of membrane potentials ex vivo when depolarized with hyperkalaemic buffers or mitochondrial uncoupling. In vivo measurements of membrane potentials agreed with literature values, whereas Δ Ψ m $\Delta {{{{\Psi}}}_{\mathrm{m}}}$ was found to be less polarized ex vivo in the perfused heart. The method uses clinically available single-photon emission computed tomography imaging agents that could be employed to measure these parameters in humans.