Sarcolemmal and mitochondrial membrane potentials measured <i>ex vivo</i> and <i>in vivo</i> in the heart by pharmacokinetic modelling of [ ^99m Tc]sestamibi

Transmembrane electrical potentials across the sarcolemmal ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:msub><mml:mi>E</mml:mi> <mml:mi>m</mml:mi></mml:msub> <mml:annotation>${{E}_{\mathrm{m}}}$</mml:annotation></mml:semantics> </mml:math> ) and mitochondrial ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mi>Δ</mml:mi> <mml:msub><mml:mi>Ψ</mml:mi> <mml:mi>m</mml:mi></mml:msub> </mml:mrow> <mml:annotation>$\Delta {{{{\Psi}}}_{\mathrm{m}}}$</mml:annotation></mml:semantics> </mml:math> ) 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 <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:msub><mml:mi>E</mml:mi> <mml:mi>m</mml:mi></mml:msub> <mml:annotation>${{E}_{\mathrm{m}}}$</mml:annotation></mml:semantics> </mml:math> and <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mi>Δ</mml:mi> <mml:msub><mml:mi>Ψ</mml:mi> <mml:mi>m</mml:mi></mml:msub> </mml:mrow> <mml:annotation>$\Delta {{{{\Psi}}}_{\mathrm{m}}}$</mml:annotation></mml:semantics> </mml:math> 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 <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics> <mml:mrow><mml:msub><mml:mi>E</mml:mi> <mml:mi>m</mml:mi></mml:msub> <mml:mo>=</mml:mo> <mml:mo>-</mml:mo> <mml:mn>65</mml:mn> <mml:mo>±</mml:mo> <mml:mn>7</mml:mn> <mml:mspace></mml:mspace> <mml:mi>mV</mml:mi></mml:mrow> <mml:annotation>${{E}_{\mathrm{m}}}=-65\pm 7\, {\mathrm{mV}} $</mml:annotation></mml:semantics> </mml:math> and <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mi>Δ</mml:mi> <mml:msub><mml:mi>Ψ</mml:mi> <mml:mi>m</mml:mi></mml:msub> <mml:mo>=</mml:mo> <mml:mo>-</mml:mo> <mml:mn>109</mml:mn> <mml:mo>±</mml:mo> <mml:mn>9</mml:mn> <mml:mspace></mml:mspace> <mml:mi>mV</mml:mi></mml:mrow> <mml:annotation>$\Delta {{{{\Psi}}}_{\mathrm{m}}}= -109 \pm 9\, {\mathrm{mV}}$</mml:annotation></mml:semantics> </mml:math> (mean ± SD, n = 4). Increasing [K⁺] caused dose-dependent depolarization of <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:msub><mml:mi>E</mml:mi> <mml:mi>m</mml:mi></mml:msub> <mml:annotation>${{E}_{\mathrm{m}}}$</mml:annotation></mml:semantics> </mml:math> in agreement with Goldman-Hodgkin-Katz predictions, whereas <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mi>Δ</mml:mi> <mml:msub><mml:mi>Ψ</mml:mi> <mml:mi>m</mml:mi></mml:msub> </mml:mrow> <mml:annotation>$\Delta {{{{\Psi}}}_{\mathrm{m}}}$</mml:annotation></mml:semantics> </mml:math> remained stable. CCCP selectively depolarized <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mi>Δ</mml:mi> <mml:msub><mml:mi>Ψ</mml:mi> <mml:mi>m</mml:mi></mml:msub> </mml:mrow> <mml:annotation>$\Delta {{{{\Psi}}}_{\mathrm{m}}}$</mml:annotation></mml:semantics> </mml:math> to <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mo>-</mml:mo> <mml:mn>66</mml:mn> <mml:mo>±</mml:mo> <mml:mn>8</mml:mn> <mml:mspace></mml:mspace> <mml:mi>mV</mml:mi></mml:mrow> <mml:annotation>$-66 \pm 8\, {\mathrm{mV}} $</mml:annotation></mml:semantics> </mml:math> (300 nm) and <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mo>-</mml:mo> <mml:mn>6</mml:mn> <mml:mo>±</mml:mo> <mml:mn>2</mml:mn> <mml:mspace></mml:mspace> <mml:mi>mV</mml:mi></mml:mrow> <mml:annotation>$-6 \pm 2\, {\mathrm{mV}} $</mml:annotation></mml:semantics> </mml:math> (600 nm) with minimal effect on <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:msub><mml:mi>E</mml:mi> <mml:mi>m</mml:mi></mml:msub> <mml:annotation>${{E}_{\mathrm{m}}}$</mml:annotation></mml:semantics> </mml:math> . In vivo, potentials were <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics> <mml:mrow><mml:msub><mml:mi>E</mml:mi> <mml:mi>m</mml:mi></mml:msub> <mml:mo>=</mml:mo> <mml:mo>-</mml:mo> <mml:mn>61</mml:mn> <mml:mo>±</mml:mo> <mml:mn>8</mml:mn> <mml:mspace></mml:mspace> <mml:mi>mV</mml:mi></mml:mrow> <mml:annotation>${{E}_{\mathrm{m}}}= -61 \pm 8\, {\mathrm{mV}}$</mml:annotation></mml:semantics> </mml:math> and <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mi>Δ</mml:mi> <mml:msub><mml:mi>Ψ</mml:mi> <mml:mi>m</mml:mi></mml:msub> <mml:mo>=</mml:mo> <mml:mo>-</mml:mo> <mml:mn>151</mml:mn> <mml:mo>±</mml:mo> <mml:mn>13</mml:mn> <mml:mspace></mml:mspace> <mml:mi>mV</mml:mi></mml:mrow> <mml:annotation>$\Delta {{{{\Psi}}}_{\mathrm{m}}}= -151 \pm 13\, {\mathrm{mV}}$</mml:annotation></mml:semantics> </mml:math> (n = 4), consistent with physiological values. This modelling approach enables the first non-invasive, independent quantitative estimation of sarcolemmal and mitochondrial membrane potentials in vivo. It overcomes limitations of optical probes and, with high-sensitivity single-photon emission computed tomography and positron emission tomography (PET) systems (including total body PET), offers new opportunities to assess bioenergetic dysfunction in cardiovascular disease and beyond. KEY POINTS: Pharmacokinetic modelling of [^99mTc]sestamibi and [^99mTc]tetrofosmin allowed independent estimation of sarcolemmal ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:msub><mml:mi>E</mml:mi> <mml:mi>m</mml:mi></mml:msub> <mml:annotation>${{E}_{\mathrm{m}}}$</mml:annotation></mml:semantics> </mml:math> ) and mitochondrial ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mi>Δ</mml:mi> <mml:msub><mml:mi>Ψ</mml:mi> <mml:mi>m</mml:mi></mml:msub> </mml:mrow> <mml:annotation>$\Delta {{{{\Psi}}}_{\mathrm{m}}}$</mml:annotation></mml:semantics> </mml:math> ) membrane potentials ex vivo in the Langendorff perfused rat heart and in vivo in the rat heart. The method gave independent measures of membrane potentials ex vivo when depolarized with hyperkalaemic buffers or mitochondrial uncoupling. In vivo measurements of membrane potentials agreed with literature values, whereas <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mi>Δ</mml:mi> <mml:msub><mml:mi>Ψ</mml:mi> <mml:mi>m</mml:mi></mml:msub> </mml:mrow> <mml:annotation>$\Delta {{{{\Psi}}}_{\mathrm{m}}}$</mml:annotation></mml:semantics> </mml:math> 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.