Quantitative in vivo mapping of myocardial mitochondrial membrane potential

PLOS ONE, Jan 2018

Background Mitochondrial membrane potential (ΔΨm) arises from normal function of the electron transport chain. Maintenance of ΔΨm within a narrow range is essential for mitochondrial function. Methods for in vivo measurement of ΔΨm do not exist. We use 18F-labeled tetraphenylphosphonium (18F-TPP+) to measure and map the total membrane potential, ΔΨT, as the sum of ΔΨm and cellular (ΔΨc) electrical potentials. Methods Eight pigs, five controls and three with a scar-like injury, were studied. Pigs were studied with a dynamic PET scanning protocol to measure 18F-TPP+ volume of distribution, VT. Fractional extracellular space (fECS) was measured in 3 pigs. We derived equations expressing ΔΨT as a function of VT and the volume-fractions of mitochondria and fECS. Seventeen segment polar maps and parametric images of ΔΨT were calculated in millivolts (mV). Results In controls, mean segmental ΔΨT = -129.4±1.4 mV (SEM). In pigs with segmental tissue injury, ΔΨT was clearly separated from control segments but variable, in the range -100 to 0 mV. The quality of ΔΨT maps was excellent, with low noise and good resolution. Measurements of ΔΨT in the left ventricle of pigs agree with previous in in-vitro measurements. Conclusions We have analyzed the factors affecting the uptake of voltage sensing tracers and developed a minimally invasive method for mapping ΔΨT in left ventricular myocardium of pigs. ΔΨT is computed in absolute units, allowing for visual and statistical comparison of individual values with normative data. These studies demonstrate the first in vivo application of quantitative mapping of total tissue membrane potential, ΔΨT.

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Quantitative in vivo mapping of myocardial mitochondrial membrane potential

January Quantitative in vivo mapping of myocardial mitochondrial membrane potential Nathaniel M. Alpert 0 1 Nicolas Guehl 0 1 Leon Ptaszek 1 Matthieu Pelletier-Galarneau 0 1 Jeremy Ruskin 1 Moussa C. Mansour 1 Dustin Wooten 0 1 Chao Ma 0 1 Kazue Takahashi 0 1 Yun Zhou 1 Timothy M. Shoup 0 1 Marc D. Normandin 0 1 Georges El Fakhri 0 1 0 Gordon Center for Medical Imaging, Massachusetts General Hospital, Harvard Medical School , Boston , Massachusetts, United States of America, 2 Cardiac Arrhythmia Service, Massachusetts General Hospital, Harvard Medical School , Boston , Massachusetts, United States of America, 3 The Russell H. Morgan Department of Radiology and Radiological Science, School of Medicine Johns Hopkins University , Baltimore, Maryland , United States of America 1 Editor: Cecilia Zazueta, Instituto Nacional de Cardiologia Ignacio Chavez , MEXICO Mitochondrial membrane potential (ΔΨm) arises from normal function of the electron transport chain. Maintenance of ΔΨm within a narrow range is essential for mitochondrial function. Methods for in vivo measurement of ΔΨm do not exist. We use 18F-labeled tetraphenylphosphonium (18F-TPP+) to measure and map the total membrane potential, ΔΨT, as the sum of ΔΨm and cellular (ΔΨc) electrical potentials. - Funding: This work was funded by two grants from the United States National Institutes of Health: R01HL110241 (GEF) and R01HL137230 (GEF), https://www.nhlbi.nih.gov. These grants were awarded as part of the standard peer review process for new grants. The study design and analysis are solely the work of the study authors, meaning that the funders had no role in study Background Methods (mV). Results Eight pigs, five controls and three with a scar-like injury, were studied. Pigs were studied with a dynamic PET scanning protocol to measure 18F-TPP+ volume of distribution, VT. Fractional extracellular space (fECS) was measured in 3 pigs. We derived equations expressing ΔΨT as a function of VT and the volume-fractions of mitochondria and fECS. Seventeen segment polar maps and parametric images of ΔΨT were calculated in millivolts In controls, mean segmental ΔΨT = -129.4±1.4 mV (SEM). In pigs with segmental tissue injury, ΔΨT was clearly separated from control segments but variable, in the range -100 to 0 mV. The quality of ΔΨT maps was excellent, with low noise and good resolution. Measurements of ΔΨT in the left ventricle of pigs agree with previous in in-vitro measurements. Conclusions We have analyzed the factors affecting the uptake of voltage sensing tracers and developed a minimally invasive method for mapping ΔΨT in left ventricular myocardium of pigs. ΔΨT is computed in absolute units, allowing for visual and statistical comparison of individual values design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. with normative data. These studies demonstrate the first in vivo application of quantitative mapping of total tissue membrane potential, ΔΨT. Introduction Mitochondria produce approximately 90% of cellular adenosine triphosphate (ATP) through oxidative phosphorylation [ 1 ]. The electron transport chain (ETC) of the mitochondrion is ultimately responsible for converting the foods we eat into electrical and chemical energy gradients by pumping protons across the inner membrane in the mitochondrial intermembrane space. The energy stored in the electric field, referred to as mitochondrial membrane potential (ΔCm), is then used to power the conversion of ADP to ATP. In a typical cell, the ΔCm remains constant with time and is about -140 mV [ 2 ]. Table 1 lists ΔCm for mitochondria of different cell types. If ΔCm remains within the physiological range, a small amount of reactive oxygen species (ROS) is produced. However, in mitochondrial dysfunction, ΔCm falls outside the normal range, with concomitant increase in ROS release, and impairment of ATP production [ 10 ]. And because mitochondria are the most important source of energy and ROS in the cell, mitochondrial dysfunction is at the core of many diseases, including myopathies [ 11 ], diabetes [ 12 ], degenerative diseases [ 13 ], inflammation [ 14 ], cancer [ 15 ], and cardiac arrhythmias [ 16, 17 ]. Despite continuing scientific interest in voltage sensitive probes, a noninvasive method for measuring ΔCm in living animals does not currently exist. The basic physiological studies conducted several decades ago are highly relevant but not always mentioned: Historically, fluorescent dyes [ 18 ] and lipophilic cationic tracers have been developed for quantitative assay of ΔCm in isolated mitochondria [ 4 ], cells [ 19 ], and isolated heart preparations [ 6 ]. Electrodes sensitive to tetraphenylphosphonium (TPP) have also been developed and used to evaluate ΔCm in isolated mitochondrial fractions [ 9, 20 ]. [14C or 3H]-labeled lipophilic cations were used to study the electrical properties of membranes and mitochondria long before modern imaging methods were imagined [3, 21±23]. More recently, Logan et al. [24] reported a "click" method for in vivo measurement of ΔC in the cells of mouse hearts, but their method requires the excision of the heart and hence is not suitable for translation to human studies. The work cited above established the use of 3H-TPP+ as a reference tracer for measuring mitochondrial membrane potential (ΔCm). Investigators have shown that 3H-TPP+ distributes 2 / 16 slowly in accord with the electrochemical gradient [ 4, 22 ]. Min et al suggested that TPP might be an important imaging agent [25].By replacing the tritium label with 18F[ 26 ], it is possible to adapt the methodology to PET, making it feasible to extend these measurements to intact animals and to human studies. In this paper, we report work that adapts the methods used in the earlier bench top measurements to in vivo imaging using positron emission tomography (PET/CT) to measure and map total membrane potential (ΔCT) We define ΔCT as the sum of ΔΨm and cellular (ΔΨc) electrical potentials. The time scale of our measurements are tens of minutes and thus ΔCc is represented by its time average. ΔCT is chosen as a practical surrogate for ΔCm, keeping in mind that in most situations ΔCm 10 ΔCc and thus ΔCT ΔCm. In our methodology a cationic lipophilic tracer TPP+, labeled with 18F, is used to quantitatively map myocardial ΔCT. 18F-TPP+ was initially developed as a myocardial flow imaging agent under the trade name BFPET [ 26 ] However, 18F-TPP+ enters the tissue with a low first-pass extraction fraction and does not respond to pharmacological challenge with a stressor and hence cannot be considered a flow tracer [ 7 ]. Nonetheless, its electrochemical properties make it a tracer of interest for quantitative imaging of ΔCT. Previous work, attempting to detect changes in concentration due to alteration of the ΔCm used tracers such as 99mTc-sestamibi [ 27 ], tetraphenyl phosphonium[ 25 ], (18F-fluoropentyl) triphenylphosphonium[ 28 ], and 18F-fluorobenzyl triphenyl phosphonium [ 29 ] [ 30 ], with semi-quantitative endpoints such as SUV [ 31 ]. The results of these studies are empirical and descriptive. In the sense that a binary decision threshold is sought; the electrical properties of transmembrane kinetics are not exploited for quantitative purposes. Changes indicative of graded mitochondrial dysfunction cannot be detected. Gurm et al reported the first attempt to quantitatively measure ΔCm with PET and 18F-TPP+ [ 7 ]. Their analysis simply applied the Nernst equation to the PET and plasma concentrations measured 30 minutes after bolus injection of 18F-TPP+. But terminating the study at 30 minutes was arbitrary and did not consider that the plasma level falls monotonically for at least 120 minutes, meaning that had they used the data at, say, 45 minutes after injection, they would have obtained a different result. Thus, their assumption that tracer was in steady state 30 minutes after bolus injection is incorrect and leads to biased results. In addition, their analysis did not consider the effect of tracer in the extracellular space. Because of these errors, their method violates basic tracer kinetic principles and their results significantly underestimate ΔCm and are not in good agreement with work from in vitro studies (Table 1). General design of the studies This investigation provides an initial assessment of a method for quantitative mapping of ΔCT. Because the mammalian heart has the highest concentration of mitochondria, we chose myocardial imaging as the first application to facilitate optimization of the scanning conditions. The subjects of our study were domestic swine (Sus domesticus) imaged in one of two conditions: healthy controls or pigs with chronic injury to the left anterior descending arterial (LAD) territory. We used bolus injection of 18F-TPP+ in control and injured pigs to measure the kinetics of TPP+ for at least two hours, determining the volume of distribution by a regression model. Methods A new method for quantification of membrane potential We partition the tissue distribution of 18F-TPP+ into several components: ECS, consisting of interstitial space and plasma, mitochondria (mito) and cytosolic (cyto) volume fractions (Fig 1). The Nernst equation [ 32, 33 ] equates transmembrane electric potential to the ratio of ion concentrations on either side of the membrane. Thus, the Nernst equation allows us to derive 3 / 16 Fig 1. Volume of distribution model for18F-TPP+ in a PET image voxel. The outer black line represents the voxel boundary. Cp, Cinter, Ccyto, and Cmito represent the concentrations of the plasma, interstitial space, cytosol, and mitochondria respectively. The arrows represent 18F-TPP+ transport between the different compartments. fECS represents the voxel volume fraction occupied by ECS and fmito represents the cellular volume fraction occupied by mitochondria. an equation relating the PET measurements of 18F-TPP+ concentration, to ΔΨT, ECS fraction (fECS), mitochondrial volume fraction (fmito) and the electric potential across the cellular membrane, ΔCc. At steady state, the concentration of 18F-TPP+ in a PET voxel can be written as CPET ˆ …1 fECS†…fmito Cmito ‡ …1 fmito† Ccyto† ‡ fECS CECS …1† where, Cmito, Ccyto and CECS are the steady state concentrations of TPP+ in the mitochondria, cytosol and ECS, respectively. At steady state plasma and ECS are equal and Cmito and Ccyto are related through the Nernst equations: CESC Ccyto ˆ e bDCc and Ccyto Cmito ˆ e bDCm 2† We divide Eq 1 by Cp and use Eq 2 to express the tracer volume of distribution, VT, as VT ˆ CPET ˆ …1 Cp fECS† fmito e bDCT ‡ …1 fmito† e bDCc ‡ fECS; …3† where b ˆ RzFT is a ratio of known physical parameters: F denotes Faraday's constant, z is the valence, R is the universal gas constant and T is the temperature in degrees Kelvin. In our calculations z = 1, F = 96485.3 [Coulombs per mole], R = 8.314472, and T = 310.2 [degrees Kelvin]. CPET is the steady-state ratio of the tissue to plasma concentrations. This equation predicts Cp that VT, a kinetically determined tracer quantity, is equal to the steady state concentration ratio CT . Thus, when the tracer concentrations in tissue and plasma are time-invariant, VT is, Cp by definition, the tissue-to-plasma concentration ratio; whereas, after bolus injection the tissue-to-plasma concentration ratio varies with time and VT must be determined kinetically. Unlike tracers whose distribution is governed by passive transport, 18F-TPP+ will have a very high (>>1) volume of distribution when the membrane potential is in the normal range. A reasonable approximation to the volume of distribution is given by VT ˆ CCTp …1 fECS† fmito e b DCT …4† Barth et al. [ 34 ] have studied 10 different mammalian species, including pigs and man, finding that the myocardial fmito is a specific and constant value for any particular species. Therefore, VT is sensitive to two independent variables; ΔΨT and fECS. The fundamental mathematical relationships expressed in Eqs 1, 2 and 3 are depicted in Fig 2, illustrating that the systematic error in ΔΨT due to neglecting fECS for normal values of VT is about 20 mV. But note that Fig 2 also shows that at low membrane potential, the effect of the ECS is negligible, meaning that knowledge of fECS is most important for detecting normal versus mildly dysfunctional mitochondria. Study cohort 18F-TPP+ scans were performed on eight Yorkshire swine (Pigs were all American Yorkshire male ordered from Animal BiowareTM Series II Sofware Suite, vendor: Tufts). There were 5 animals without tissue injury, referred to as control pigs, and 3 animals with a left anterior descending artery (LAD) infarction, referred to as injury pigs. Pigs shared a room with other animals of the same species, individually housed in their own cage, free to turn and make normal movements and postural adjustments. The animals were given play toys to enrich their environment and fed adequately, with ready access to water, to ensure normal growth. An acclimation period greater than 4 days was observed upon the animal's arrival before conducting the first imaging procedure. Animals used in this study were housed and maintained under the supervision of the Massachusetts General Hospital Animal Care and Use Committee and our study was conducted under a protocol approved by the Institutional Animal Care and Use Committee of the Massachusetts General Hospital. Anesthesia Following a 12 hour fast, pigs were sedated with 4.4 mg/kg Telazol. Anesthesia was induced with isoflurane 5% and maintained with 1.5% isoflurane. During anesthesia, the animals were 5 / 16 Fig 2. Dependence of VT on mitochondrial membrane potential and fractional ECS computed with Eq 3. ΔT is more negative inside membranes. Black lines indicate variation (systematic error) in ΔT range at constant volume of distribution due to neglecting fractional ECS volume. mechanically ventilated. Vital signs and depth of anesthesia were assessed once every 5 minutes and this assessment was documented every 15 minutes. Tissue injury Percutaneous central access was achieved via the Seldinger technique [ 35 ] under anesthesia. Femoral artery access was obtained and a guide wire was advanced into the LAD coronary artery. A balloon catheter was fed over the guidewire, placed in the mid LAD, and inflated to 6±8 atm for 80 minutes. An infarct was confirmed by the appearance of large ST elevation on ECG. At the end of the surgical procedure, subcutaneous Carprofen was administered and the animal was returned to housing for recuperation. After the immediate postoperative period, the animals were observed at least twice daily by study staff. Post-procedural Carprofen was administered orally for 3 days at a dose of 150 mg/day and then as needed if stiffness or swelling continued after that point. In order to prevent arrhythmias, 200mg Amiodarone and 50 mg Atenolol were also given orally every day during 2 weeks. During the study, animal health and well-being, as well as the adequacy of anesthesia, were monitored by checking respiration rate, ECG, blood gas, corneal or palpebral reflex, blood pressure, heart rate, pulse oximetry. Blood sampling Radiotracer injection, iodinated contrast injection, gadolinium injection, and venous blood sampling were performed through femoral vein catheters. An arteriovenous shunt was placed in the left femoral artery for arterial blood sampling. After tracer injection, arterial blood 6 / 16 samples were obtained every 10 seconds for the first 3 minutes, then at 1 minute intervals for 5 minutes, and at increasing intervals until 120 minutes post injection. Venous blood samples were obtained at 5, 10, 15, 30, 60 and 90 minutes after tracer injection. All blood samples were centrifuged to determine plasma and red cell concentration histories. Scanning Eight scans, 5 control and 3 injury pigs, were performed using a Siemens Biograph 64 PET/ CT. Following administration of 185 MBq of 18F-TPP+ as a single intravenous bolus, scanning was performed over 120 minutes in list mode. CT angiography was performed for anatomic reference. List mode data were framed as a dynamic series of 12x3, 9x5, 7x10, 15x30 second frames. PET/CT data were reconstructed using a filtered back projection algorithm with CTbased attenuation correction to yield a radioactivity concentration map in units of Bq/cc with 83 slices and a voxel size of 2.14x2.14x3 mm3. fECS was measured in 3 injury pigs with CT scanning using a bolus plus infusion iodinated contrast protocol [ 36 ]. Data analysis Dynamic PET data were analyzed using a Logan regression method [ 37 ] to produce quantitative parametric images of the VT of 18F-TPP+. Images of VT were reoriented into the short axis projection and cropped so that the cardiac chambers occupied nearly all of the image space. Eq 3 was used to convert the parametric maps of VT to maps of ΔCT, assuming ΔCc = -15 mV [ 7 ] and fmito = 0.26 [ 34 ]. Segmental values are reported as a grand mean ΔCT and its standard error of the mean (SEM). No background subtraction or thresholding was applied to the parametric images of VT. Results In vivo mapping of ΔΨT Fig 2 was computed using Eq 3 to show the predicted behavior of mitochondrial membrane potential as a function of total volume of distribution, and size of the extracellular space. ΔCc was assumed to be -15 mV for these calculations. Fig 2 shows a nearly exponential behavior and that ΔΨT is nearly independent of fECS when membrane potential drops below about -100 mV. As shown in Fig 3, after bolus injection, the plasma concentration history decreased rapidly during the "equilibration" phase and slowly thereafter; whereas, TPP demonstrated an extended myocardial residence time characterized by nearly constant or slowly decreasing tissue concentration. The concentration ratio for whole blood versus plasma became constant, with a mean value of 0.98±0.02 (SEM), about 15 minutes after injection of TPP+ (Fig 4). Venous and arterial samples obtained later than 10 minutes after injection of TPP+ were in good agreement. 18F-TPP+ concentration was highest in heart and liver, reaching a plateau in normal myocardium after about 10 minutes and declining very slowly thereafter. The uptake of 18F-TPP+ in scar was variable, reflecting the variation in degree and extent of injury. Tissue concentration in the injured area peaked about 10 minutes after injection, followed by a slow biphasic clearance, with the plateau level about 40% as high as in the normal myocardium. In normal left ventricular myocardium, average fECS = 0.20 and varied less than 10%. Local values of fECS could not be obtained in the tissue injury, due to poor signal-to-noise ratio in the CT-studies and average values were used in computation of ΔΨT. 7 / 16 Fig 3. Variation of plasma and tissue concentration following intravenous bolus injection of 18F-TPP+. Plasma concentration decreases monotonically over the first two hours; whereas, myocardial concentration is nearly constant. TPP+ is avidly taken up by normal LV myocardium, as shown in Fig 5. The distribution of 18F-TPP+ is shown as SUV integrated from 60±120 min post tracer injection in the three oblique projections obtained directly from the PET image volume. No further processing was done to these images. Representative parametric images of VT and ΔΨT derived from kinetic data obtained from a control pig and an injury pig were reoriented into the standard cardiac coordinate system and presented in Fig 6. Images of VT have units of cc-tissue/g-plasma; whereas, images of ΔΨT are in units of negative millivolts (-mV). The apparent lower volume of distribution in right ventricle and atria is artifactual, due to the thinner walls of those structures in combination with the effects of finite spatial resolution and cardiac motion blurring. Segmental ΔΨT is tightly grouped for the five control pigs with a grand mean ± SEM over 17 segments of -129.4±1.4 mV (Fig 7). Values of ΔΨT are lower in injured segments, particularly in the apical-septal segments corresponding to the injury in the territory of the LAD artery. 8 / 16 Fig 4. Whole blood-to-plasma concentration ratio measured as a function of time after bolus injection of 18F-TPP+. After about 15 minutes, whole blood and plasma concentrations equilibrate with equal concentration. Discussion In this paper, we introduce the concept of quantitative mapping of ΔΨT for monitoring mitochondrial status. Our development emphasizes measurement of the total membrane potential, ΔΨT, while making clear that ΔΨT is a proxy for and tightly correlated to ΔΨm. Direct measurements of ΔΨm require a separate measurement of the cellular membrane potential that is currently not possible in vivo. Just as in the early studies conducted with 3H-TPP+, our method relies on measuring the total concentration of a lipophilic cationic tracer which is then analyzed by using a compartment model of the tissue and the steady state formulation of the Fig 5. Typical 18F-TPP+ SUV image, integrated from 60±120 minutes after IV bolus injection. SUV is highest in liver, followed by LV myocardium, with lower activity in the visible in bone marrow. 9 / 16 Fig 6. Parametric images of VT and ΔΨT. Each panel of three x two images shows short axis, vertical and horizontal slices. Images of a representative control pig are shown in the left panel. Images of a representative scar pig are shown in the right panel. The top row of each panel depicts the TPP+ volume of distribution and bottom row the membrane potential. Nernst equation, the same equation that is fundamental to electrochemistry [ 38 ] and cardiac electrophysiology [ 39, 40 ]. The relation of tracer concentration, which varies, and the physiological steady state can seem confusing. In the physiological state all the biological concentrations, transport rates and electropotentials are assumed to be fixed; whereas, the tracer concentrations evolve over time during the experiment (Fig 3). This complicates the application of the Nernst equation, whose use assumes the concentrations determining the membrane potential are invariant with time. We addressed that issue by using a kinetic model with extracellular and intracellular pools measured with dynamic PET to estimate the total volume of tracer distribution, a time invariant quantity characteristic of the tracer and the animal under study. We explicitly considered the effect of the electric field across the inner membrane of the mitochondrion on the kinetics Fig 7. Comparison of ΔT in 17 "bull's eye" segments Results shown for five control (blue squares) and three pigs with injury to the LAD territory (red circles). 10 / 16 of TPP+ by using a kinetic model to express the relation between the total volume of distribution of the tracer and the electrical properties of the membrane. The structure of this model is identical to that used to analyze the classic 3H-TPP+ experiments conducted many years ago. While qualitative imaging may serve many purposes, the quantitative aspects of our method may provide additional important information about mitochondrial status not available from visual inspection of images, true because mitochondrial dysfunction may, in some tissues, be associated with uniformly reduced ΔΨm and such conditions cannot be detected by qualitative imaging methods. Another potentially important result of this work is the more analytic understanding of the factors affecting the distribution of TPP+, in particular, and voltage sensing tracers in general. We used basic principles of physics and physiology to show how the volume of distribution of lipophilic cations depends on the volume fractions of the tissue occupied by the ECSand the mitochondria as well as on the magnitude of ΔCT. VT is approximately equal to …1 fECS† fmito e DCT . Keeping in mind that for normal tissue the mitochondrial membrane potential is about -140 mV [ 2 ] for all mitochondria regardless of tissue type; whereas, the fractional mitochondrial volume varies by more than a factor 10, Eqs 3 and 4 imply that the intensity of the VT image will reflect the mitochondrial volume fraction of the tissue, thereby explaining the intensity variations depicted in Fig 5. Eqs 3 and 4 also make clear that the size of the ECS is an important factor when interpreting TPP+ images because this quantity may vary with age and disease. Fig 2 shows the model-prediction for the volume of distribution when we fix the mitochondrial volume fraction while varying the membrane potential and the fractional volume of the ECS. This result shows that increases in the size of the ECShave decreasing effect on the total volume of distribution as the membrane potential decreases and becomes depolarized. But if the goal is to detect more subtle changes from normal ΔCT it is important to include the effect of variation in ECS. Failure to account for changes in ECS may lead to unexplained variability in the qualitative and quantitative assessments of voltage-sensing tracer distribution. ΔΨm is sustained by the electron transport chain of the mitochondria, by which a balance is struck between protons pumped across the inner mitochondrial membrane and those pumped back to power the synthesis of ATP. ΔΨm is also affected by changes in the level of ROS and various mitochondrial ion channels [ 41 ]. Interestingly, increased ROS levels and modulation of mitochondrial ion channel function are seen early in numerous pathologies. Thus, the ability to quantitatively map ΔΨT may be useful for diagnosing or managing a number of such conditions. For example, reduction in ΔΨm has been implicated as a mechanism underlying ventricular arrhythmogenesis [ 42 ] and so might be used to improve the detection of arrhythmogenic foci. We noted in Introduction that there are currently no methods for measuring ΔΨm in animal or human. This means that there is no independent method against which we can compare our PET methods. In this regard, it is important to emphasize that our noninvasive PET method is strongly related to the highly invasive and validated bench-top methods which preceded it. All prior methods employing in vitro application of cationic tracers were based on the Nernst equation to relate steady state concentrations and membrane potential in a compartment model of the mitochondrion, cell or tissue. Similarities to PET can be seen with 3H-TPP+ studies conducted in populations of cells and isolated mitochondria, where concentrations of TPP+ in the medium are related to the concentration in ensembles of cells or mitochondria by the Nernst equation. Measurements with TPP+ electrodes are also based on the same principles [ 9 ]. The similarity is most apparent in the work of Wan et al. [ 6 ] who studied ΔΨm in isolated rat hearts by using the arterio-venous difference in concentration of 3H-TPP+ 11 / 16 to infer the tissue-to-perfusate concentration ratio. In essence, measuring the evolution of 18F-TPP+ concentration in a PET ROI is completely analogous to measurements with 3H-TPP+ in the isolated perfused rat heart studies of Wan et al. [ 6 ]. Hence, the PET studies are the natural extension of the classical bench top reference methods. The fact that our PET measurements of ΔΨT are in accord with measurements in isolated rat hearts is an important observation, especially since direct validation of our method is impossible with existing methodologies. As illustrated in Table 1, the PET method yields results that are close to those found by the majority of prior studies, thereby providing additional support of our method. With high specific activity, the mass of 18F-TPP+ is 10−10 to 10−7 lower than the intracellular potassium concentration, meaning its effect on membrane potential is negligible. However, it is worth mentioning that prior studies with 3H-TPP+ have sometimes made corrections for non-specific binding of TPP+ [ 4 ] but the literature is not concordant on the necessity of correction [4, 9, 43±45]. Furthermore, previous bench top studies used very low specific activity TPP+, complicating an evaluation of its necessity in tracer measurements. Nevertheless, similar corrections could be applied to the PET analyses, but were found to be unnecessary to establish initial validity. Close inspection of Fig 5 shows there is a high value of VT in normal myocardium, with mitochondrial concentrations nearly 30 times the plasma level. At secular equilibrium, the inward and outward fluxes across the mitochondrial membrane have to balance, meaning that outward clearance must be about 30 times lower than the inward rate, thereby explaining the slow clearance observed experimentally after bolus injection of 18F-TPP+. Fig 5 also shows that the volume of distribution in injured myocardium is much lower, with tissue concentrations less than 10 times the plasma concentration. Our kinetic model shows that VT depends linearly on fECS and exponentially on ΔΨT. Accordingly, we have converted VT to an estimate of ΔΨT by accounting for the effects of fECS, a quantity known to vary in age and disease [46±49]. Thus, we have shown it will be necessary to account for changes in extracellular volume fraction in clinical studies to obtain the full benefit of such studies. A marked difference in contrast between VT and ΔΨT was observed between normal and injured tissue (Fig 5). Both VT and ΔΨT are quantitative measures emphasizing different aspects of 18F-TPP+ distribution. On one hand, VT is the total volume of 18F-TPP+ distribution, including effects due to ΔΨT, fECS, and fmito. Therefore, mitochondrial density (fmito) will affect the relative uptake of a voltage-sensing tracer. This finding is interesting given that different pathologies are associated with reduced mitochondrial density. For example, a reduction in mitochondrial density is seen in the skeletal muscles of patients with chronic obstructive pulmonary disease [ 50 ]. In these conditions, VT measurements would provide an overall quantitative measure of mitochondrial status in tissue. On the other hand, ΔΨT focuses predominantly on the electrochemical conditions that prevail at the inner mitochondrial membrane. In the large tissue injury, we see that there is an area of profound reduction in ΔΨT, approaching total depolarization. Other parts of the injured region show lesser depolarization in the range of -60 mV, but still very different than ΔΨT in normal myocardium. This can also be appreciated from the data in Fig 7, where the blue circles indicate major reductions in the average ΔΨT for bull's eye segment 8, 9, 14, 15 and 17. We can also see the variability of tissue injury expressed in Fig 7, which demonstrate a patchy nature to those injuries that might be better appreciated by direct examination of the parametric images. As also shown in Fig 7, pigs in the control group exhibited segmental values of ΔΨT that averaged about -129.4 ± 1.4 mV (SEM). The tight grouping of ΔΨT measurements over all normal segments and pigs is remarkable. The kinetic approach, used in this study, requires a long measurement period for accurate estimation of the total volume of distribution. A protocol 12 / 16 using primed constant infusion may be preferred for human investigation in order to restrict actual scan time to the equilibrium period, 95±120 min. In this work we reported the results of kinetic analysis using the Logan graphical method, which is known to underestimate VT with increasing statistical noise in the measurements[ 51 ]. We mitigated the effect of statistical noise by averaging ΔΨT maps for 17 polar segments but the reader should be aware that the spatial averaging also causes some underestimation of ΔΨT. We also formed parametric images using the reGP method of Zhou et al [ 52 ] and the total least squares approach of Varga and Szabo,[ 53 ] but these methods yielded noisy parametric images; overall, they were not an improvement over the Logan plot. Conclusions This study is the first to demonstrate the feasibility of quantitative in vivo mapping of total membrane potential, ΔΨT, a proxy of ΔΨm. In vivo measurements of ΔΨT obtained with our new method yielded values remarkably constant within and across the hearts of domestic swine that are comparable to results from in vitro bench top experiments. We have derived a theory explaining, for the first time, the major factors affecting the transport and residence time of lipophilic cations in tissue, including ΔΨT and fECS. The fact that we can measure ΔCT in mV suggests that it may be possible to compare individual's studies with normative data. Given the critical role of mitochondrial function in numerous pathologies, the potential applications of this new imaging method are immense. This novel technique could eventually be proved useful in numerous clinical and research scenarios. Acknowledgments The authors thank Kevin Cordaro, Victoria Douglas and Julia Scotton for animal preparation, handling and monitoring. We are also grateful to Dr. Moses Wilks and Dr. Eline Verwer for their help during experimental measurements. We also thank Henry Gewirtz, M.D. for helpful discussions. Author Contributions Conceptualization: Nathaniel M. Alpert, Moussa C. Mansour, Yun Zhou, Timothy M. Shoup. Data curation: Nicolas Guehl. Formal analysis: Nathaniel M. Alpert, Nicolas Guehl. Funding acquisition: Georges El Fakhri. Investigation: Nathaniel M. Alpert, Leon Ptaszek, Matthieu Pelletier-Galarneau, Dustin Wooten, Chao Ma, Kazue Takahashi, Georges El Fakhri. Methodology: Nathaniel M. Alpert, Nicolas Guehl, Leon Ptaszek, Jeremy Ruskin, Moussa C. Mansour, Dustin Wooten, Chao Ma, Kazue Takahashi, Yun Zhou, Timothy M. Shoup, Marc D. Normandin, Georges El Fakhri. Project administration: Georges El Fakhri. Resources: Jeremy Ruskin, Moussa C. Mansour. 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Nathaniel M. Alpert, Nicolas Guehl, Leon Ptaszek, Matthieu Pelletier-Galarneau, Jeremy Ruskin, Moussa C. Mansour, Dustin Wooten, Chao Ma, Kazue Takahashi, Yun Zhou, Timothy M. Shoup, Marc D. Normandin, Georges El Fakhri. Quantitative in vivo mapping of myocardial mitochondrial membrane potential, PLOS ONE, 2018, DOI: 10.1371/journal.pone.0190968