Membrane Potential Imaging Research Articles

Calorie restriction modulates mitochondrial dynamics and autophagy in leukocytes of patients with obesity

Although it is established that caloric restriction offers metabolic and clinical benefits, the molecular mechanisms underlying these effects remain unclear. Thus, this study aimed to investigate whether caloric restriction can modulate mitochondrial function and remodelling and stimulate autophagic flux in the PBMCs of patients with obesity. This was an interventional study of 38 obese subjects (BMI > 35 kg/m2) who underwent 6 months of dietary therapy, including a 6-week very-low-calorie diet (VLCD) followed by an 18-week low-calorie diet (LCD). We determined clinical variables, mitochondrial function parameters (by fluorescence imaging of mitochondrial ROS and membrane potential), and protein expression of markers of mitochondrial dynamics (MNF1, MFN2, OPA, DRP1 and FIS1) and autophagy (LC3, Beclin, BCL2 and NBR1) by western blot. Caloric restriction induced an improvement in metabolic outcomes that was accompanied by an increase in AMPK expression, a decrease of mitochondrial ROS and mitochondrial membrane potential, which was associated with increased markers of mitochondrial dynamics (MFN2, DRP1 and FIS1) and activation of autophagy as evidenced by augmented LC3 II/I, Beclin1 and NBR1, and a decrease in BCL2. These findings shed light on the specific molecular mechanisms by which caloric restriction facilitates metabolic improvements, highlighting the relevance of pathways involving energy homeostasis and cell recovery, including mitochondrial function and dynamics and autophagy.

Imaging of intracellular mitochondrial membrane potential with a highly photostable NIR fluorescent probe

Mitochondrial membrane potential (ΔΨm) participated in important physiological processes such as ATP synthesis, ion exchange, and plays an important role in maintaining normal life activities of cells. Herein, we synthesized a fluorescent probe EDXB (3-ethyl-2-(2-(6-methoxy-2,3-dihydro-1H-xanthen-4-yl) vinyl) benzothiazol-3-ium iodide) with positive charge, which targets mitochondria by electrostatic attraction. The probe has twisted intramolecular charge transfer (TICT) characteristics, resulting in near-infrared fluorescence emission enhanced with the increase of viscosity, when the probe falls off from the mitochondrial membrane to the low-viscosity cytoplasmic aqueous phase, the fluorescence emission intensity is weakened. Therefore, the EDXB exhibited favorable targeting. In addition, EDXB also has good anti-bleaching and pH stability, which enables it to accurately feedback the change of ΔΨm. In the experiment, the intracellular imaging of EDXB certificated mitochondrial depolarization (decrease of ΔΨm) during various cell injury events including cell inflammation, apoptosis and mitophagy. We expect EDXB to be a useful tool for the analysis of ΔΨm changes.

The recruitment of mechanosensitive enteric neurons in the guinea pig gastric fundus is dependent on ganglionic stretch level.

Serving as a reservoir, the gastric fundus can expand significantly, with an initial receptive and a following adaptive relaxation, controlled by extrinsic and intrinsic reflex circuits, respectively. We hypothesize that mechanosensitive enteric neurons (MEN) are involved in the adaptive relaxation, which is initiated when a particular gastric volume and a certain stretch of the stomach wall is reached. To investigate whether the responsiveness of MEN in the gastric fundus is dependent on tissue stretch, we performed mechanical stimulations in stretched versus ganglia "at rest". Responses of myenteric neurons in the guinea pig gastric fundus were recorded with membrane potential imaging using Di-8-ANEPPS. MEN were identified by small-volume intraganglionic injection in ganglia stretched to different degrees using a self-constructed stretching tool. Immunohistochemical staining identified the neurochemical phenotype of MEN. Hexamethonium and capsaicin were added to test their effect on recruited MEN. In stretched compared to "at rest" ganglia, significantly more MEN were activated. The change in the ganglionic area correlated significantly with the number of additional recruited MEN. The additional recruitment of MEN was independent from nicotinic transmission and the ratio of active MEN in stretched ganglia shifted towards a nitrergic phenotype. The higher number of active MEN with increasing stretch of the ganglia and their greater share of nitrergic phenotype might indicate their contribution to the adaptive relaxation. Further experiments are necessary to address the receptors involved in mechanotransduction.

Analysis of the effect of the scorpion toxin AaH-II on action potential generation in the axon initial segment

The toxin AaH-II, from the scorpion Androctonus australis Hector venom, is a 64 amino acid peptide that targets voltage-gated Na+ channels (VGNCs) and slows their inactivation. While at macroscopic cellular level AaH-II prolongs the action potential (AP), a functional analysis of the effect of the toxin in the axon initial segment (AIS), where VGNCs are highly expressed, was never performed so far. Here, we report an original analysis of the effect of AaH-II on the AP generation in the AIS of neocortical layer-5 pyramidal neurons from mouse brain slices. After determining that AaH-II does not discriminate between Nav1.2 and Nav1.6, i.e. between the two VGNC isoforms expressed in this neuron, we established that 7 nM was the smallest toxin concentration producing a minimal detectable deformation of the somatic AP after local delivery of the toxin. Using membrane potential imaging, we found that, at this minimal concentration, AaH-II substantially widened the AP in the AIS. Using ultrafast Na+ imaging, we found that local application of 7 nM AaH-II caused a large increase in the slower component of the Na+ influx in the AIS. Finally, using ultrafast Ca2+ imaging, we observed that 7 nM AaH-II produces a spurious slow Ca2+ influx via Ca2+-permeable VGNCs. Molecules targeting VGNCs, including peptides, are proposed as potential therapeutic tools. Thus, the present analysis in the AIS can be considered a general proof-of-principle on how high-resolution imaging techniques can disclose drug effects that cannot be observed when tested at the macroscopic level.

Mechanosensitive enteric neurons in the guinea pig gastric fundus and antrum.

Coping with the ingested food, the gastric regions of fundus, corpus, and antrum display different motility patterns. Intrinsic components of such patterns involving mechanosensitive enteric neurons (MEN) have been described in the guinea pig gastric corpus but are poorly understood in the fundus and antrum. To elucidate mechanosensitive properties of myenteric neurons in the gastric fundus and antrum, membrane potential imaging using Di-8-ANEPPS was applied. A small-volume injection led to neuronal compression. We analyzed the number of MEN and their firing frequency in addition to the involvement of selected mechanoreceptors. To characterize the neurochemical phenotype of MEN, we performed immunohistochemistry. In the gastric fundus, 16% of the neurons reproducibly responded to mechanical stimulation and thus were MEN. Of those, 83% were cholinergic and 19% nitrergic. In the antrum, 6% of the neurons responded to the compression stimulus, equally distributed among cholinergic and nitrergic MEN. Defunctionalizing the sensory extrinsic afferents led to a significant drop in the number of MEN in both regions. We provided evidence for MEN in the gastric fundus and antrum and further investigated mechanoreceptors. However, the proportions of the chemical phenotypes of the MEN differed significantly between both regions. Further investigations of synaptic connections of MEN are crucial to understand the hardwired neuronal circuits in the stomach.

Supertemporal Resolution Imaging of Membrane Potential via Stroboscopic Microscopy.

Membrane potential and its fluctuation are fundamental biophysical phenomena essential to cellular activities and functions. Compared to traditional electrode-based techniques, the optical recording via developed genetically encoded voltage indicators (GEVIs) offers a combination of noninvasiveness, high spatial resolution, and increased measurement throughput. However, its application is limited by the insufficient acquisition rate and time accuracy of the camera. Here we design and apply a stroboscopic illumination scheme to boost the temporal resolution of voltage imaging, while simultaneously eliminating the artifacts caused by nonsynchronized exposure in the rolling-shutter mode. We demonstrate that commonly used GEVIs are compatible with stroboscopic voltage imaging (SVI), and our SVI scheme offers a 5-fold faster acquisition frame rate than that of conventional continuous illumination. The GEVIs tested maintain high sensitivities in the SVI mode, supporting faithful reports of intracellular depolarization waveform and intercellular gap junction-mediated depolarization coupling in human embryonic kidney 293T (HEK 293T) cell populations. SVI allows resolving the action potential (AP) waveform with less distortion and mapping action potential initiation and propagation dynamics in cultured neurons in kilohertz, beyond the restriction from the camera in the field of view.

Control of the Electroporation Efficiency of Nanosecond Pulses by Swinging the Electric Field Vector Direction.

Reversing the pulse polarity, i.e., changing the electric field direction by 180°, inhibits electroporation and electrostimulation by nanosecond electric pulses (nsEPs). This feature, known as "bipolar cancellation," enables selective remote targeting with nsEPs and reduces the neuromuscular side effects of ablation therapies. We analyzed the biophysical mechanisms and measured how cancellation weakens and is replaced by facilitation when nsEPs are applied from different directions at angles from 0 to 180°. Monolayers of endothelial cells were electroporated by a train of five pulses (600 ns) or five paired pulses (600 + 600 ns) applied at 1 Hz or 833 kHz. Reversing the electric field in the pairs (180° direction change) caused 2-fold (1 Hz) or 20-fold (833 kHz) weaker electroporation than the train of single nsEPs. Reducing the angle between pulse directions in the pairs weakened cancellation and replaced it with facilitation at angles <160° (1 Hz) and <130° (833 kHz). Facilitation plateaued at about three-fold stronger electroporation compared to single pulses at 90-100° angle for both nsEP frequencies. The profound dependence of the efficiency on the angle enables novel protocols for highly selective focal electroporation at one electrode in a three-electrode array while avoiding effects at the other electrodes. Nanosecond-resolution imaging of cell membrane potential was used to link the selectivity to charging kinetics by co- and counter-directional nsEPs.

Assessment of Tissue Viability by Functional Imaging of Membrane Potential.

Electrical activity plays a key role in physiology, in particular for signaling and coordination. Cellular electrophysiology is often studied with micropipette-based techniques such as patch clamp and sharp electrodes, but for measurements at the tissue or organ scale, more integrated approaches are needed. Epifluorescence imaging of voltage-sensitive dyes ("optical mapping") is a tissue non-destructive approach to obtain insight into electrophysiology with high spatiotemporal resolution. Optical mapping has primarily been applied to excitable organs, especially the heart and brain. Action potential durations, conduction patterns, and conduction velocities can be determined from the recordings, providing information about electrophysiological mechanisms, including factors such as effects of pharmacological interventions, ion channel mutations, or tissue remodeling. Here, we describe the process for optical mapping of Langendorff-perfused mouse hearts, highlighting potential issues and key considerations.

Fluorinated triphenylphosphonium analogs improve cell selectivity and invivo detection of mito-metformin.

Triphenylphosphonium (TPP+) conjugated compounds selectively target cancercells by exploiting their hyperpolarized mitochondrial membrane potential. Todate, studies have focused on modifying either the linker or the cargo of TPP+-conjugated compounds. Here, we investigated the biological effects ofdirect modification to TPP+ to improve the efficacy and detection of mito-metformin (MMe), a TPP+-conjugated probe we have shown to have promising preclinical efficacy against solid cancer cells. We designed, synthesized, and tested trifluoromethyl and methoxy MMe analogs (pCF3-MMe, mCF3-MMe, and pMeO-MMe) against multiple distinct human cancer cells. pCF3-MMe showed enhanced selectivity toward cancer cells compared to MMe, while retaining thesame signaling mechanism. Importantly, pCF3-MMe allowed quantitative monitoring of cellular accumulation via 19F-NMR invitro and invivo. Furthermore, adding trifluoromethyl groups to TPP+ reduced toxicity invivo while retaining anti-tumor efficacy, opening an avenue to de-risk these next-generation TPP+-conjugated compounds.

In vivo metabolic imaging identifies lipid vulnerability in a preclinical model of Her2+/Neu breast cancer residual disease and recurrence.

Recurrent cancer cells that evade therapy is a leading cause of death in breast cancer patients. This risk is high for women showing an overexpression of human epidermal growth factor receptor 2 (Her2). Cells that persist can rely on different substrates for energy production relative to their primary tumor counterpart. Here, we characterize metabolic reprogramming related to tumor dormancy and recurrence in a doxycycline-induced Her2+/Neu model of breast cancer with varying times to recurrence using longitudinal fluorescence microscopy. Glucose uptake (2-NBDG) and mitochondrial membrane potential (TMRE) imaging metabolically phenotype mammary tumors as they transition to regression, dormancy, and recurrence. “Fast-recurrence” tumors (time to recurrence ~55 days), transition from glycolysis to mitochondrial metabolism during regression and this persists upon recurrence. “Slow-recurrence” tumors (time to recurrence ~100 days) rely on both glycolysis and mitochondrial metabolism during recurrence. The increase in mitochondrial activity in fast-recurrence tumors is attributed to a switch from glucose to fatty acids as the primary energy source for mitochondrial metabolism. Consequently, when fast-recurrence tumors receive treatment with a fatty acid inhibitor, Etomoxir, tumors report an increase in glucose uptake and lipid synthesis during regression. Treatment with Etomoxir ultimately prolongs survival. We show that metabolic reprogramming reports on tumor recurrence characteristics, particularly at time points that are essential for actionable targets. The temporal characteristics of metabolic reprogramming will be critical in determining the use of an appropriate timing for potential therapies; namely, the notion that metabolic-targeted inhibition during regression reports long-term therapeutic benefit.

Membrane-Activated Fluorescent Probe for High-Fidelity Imaging of Mitochondrial Membrane Potential.

Mitochondrial membrane potential (ΔΨm) is a key indicator of cell health or injury due to its vital roles in adenosine 5'-triphosphate synthesis. Thus, monitoring ΔΨm is of great significance for the assessment of cell status, diagnosis of diseases, and medicament screening. Cationic fluorescent probes suffer from severe photobleaching or false positive signals due to the luminescence of the probe on non-mitochondria. Herein, we report a lipophilic cationic fluorescent probe [1-methyl-2-(4-(1,2,2-triphenylvinyl)styryl)-β-naphthothiazol-1-ium trifluoromethanesulfonate (TPE-NT)] with the features of aggregation-induced emission and intramolecular charge transfer for imaging ΔΨm in live cells. TPE-NT is enriched on the surface of the mitochondrial inner membrane due to the negative ΔΨm, and its fluorescence is activated in the high-viscosity microenvironment. The false positive signals of emission from TPE-NT on non-mitochondria are therefore effectively eliminated. Moreover, TPE-NT exhibits a Stokes shift of >200 nm, near-infrared (∼675 nm) emission, excellent photostability, and low cytotoxicity, which facilitate real-time imaging in live cells. Cell imaging confirmed that the probe can rapidly and reliably report mitochondrial depolarization (decrement of ΔΨm) during cell damage caused by CCCP and H2O2 as well as mitochondrial polarization (increment of ΔΨm) by oligomycin. Furthermore, the probe successfully detected the reduction of ΔΨm in these cell models of hypoxia, heat damage, acidification, aging, inflammation, mitophagy, and apoptosis caused by hypoxia, heatstroke, lactate/pyruvate, doxorubicin, lipopolysaccharide, rapamycin, monensin, and nystatin, respectively.

Febrile Temperature Reprograms the Metabolic Phenotype in Monocyte-Derived Dendritic Cells

Fever-like hyperthermia is known to stimulate innate and adaptive immune responses. Although pyrogenic cytokines can stage the ground for immune defense (e.g. during the mobilization of lymphocytes to the lymphoid organs) hyperthermia per se is able to activate immune-competent cells and to induce a specific immunophenotype and gene expression profile. Hyperthermia-induced immune stimulation is also accompanied with, and likely conditioned by, changes in the cell metabolism and, in particular, mitochondrial metabolism is now recognized to play a pivotal role in this context, both as energy supplier and as signaling platform. In this study we asked if challenging human monocyte-derived dendritic cells with a relatively short-time thermal shock in the fever-range, typically observed in humans, caused alterations in the mitochondrial oxidative metabolism. We found that following hyperthermic stress (3 h exposure at 39°C) TNF-alpha-releasing dendritic cells undergo rewiring of the oxidative metabolism hallmarked by decrease of the mitochondrial respiratory activity and of the oxidative phosphorylation and increase of lactate production. Moreover, enhanced production of reactive oxygen and nitrogen species and accumulation of mitochondrial Ca2+ was consistently observed in hyperthermia-conditioned dendritic cells and exhibited a reciprocal interplay. The hyperthermia-induced impairment of the mitochondrial respiratory activity was (i) irreversible following re-conditioning of cells to normothermia, (ii) mimicked by exposing normothermic cells to the conditioned medium of the hyperthermia-challenged cells, (iii) largely prevented by antioxidant and inhibitors of the nitric oxide synthase and of the mitochondrial calcium porter, which also inhibited release of TNF-alpha. These observations combined with gene expression analysis support a model based on a thermally induced autocrine signaling, which rewires and sets a metabolism checkpoint linked to immune activation of dendritic cells.Figure Legend.Fig. 1A Effect of mild hyperthermic treatment of MoDCs on reactive oxygen species production in live cells and respiratory activityFig. 1B Imaging of mitochondrial membrane potential in live cells and measurement of Complex V activityFig. 1C Effect of mild hyperthermic treatment of MoDCs on the activities of specific segments of the mitochondrial respiratory chainFig. 1D Working model of the effect of mild hyperthermic conditioning of MoDCs on cell metabolism and pro-inflammatory activation [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Fabrication of a Nanoplasmonic Chip to Enhance Neuron Membrane Potential Imaging by Metal-Enhanced Fluorescence Effect

Optical imaging is a useful tool to acquire neural activities because of its high spatial resolution, and various voltage indicators were developed to image membrane potential of neurons. Voltage sensitive dyes (VSDs) are one of them but their signal-to-noise ratio (SNR) is so low that enhancing SNR of VSD has become important. In this study, we investigated the metal-enhanced fluorescence (MEF) effect on VSD imaging by fabricating nanoplasmonic resonance chip using gold nanorods (GNRs). To amplify the fluorescence signal we used polyelectrolyte layers to control the distance between metal nanoparticles and fluorophore. Cultured rat hippocampal neurons and di-8-ANEPPS, a widely used VSD, were used to test the nanoplasmonic resonance chip, and the maximum level of fluorescence signal was obtained when nine layers of polyelectrolyte spacer were used. The nanoplasmonic resonance chip with GNR showed the possibility of the improvement in voltage imaging of neurons and is expected to enhance the availability of neuronal activity imaging in the future.

Membrane Potential Imaging with Voltage-sensitive Dye (VSD) for Long-term Recording

Membrane Potential Imaging with Voltage-sensitive Dye (VSD) for Long-term Recording

Recent Progress of Hybrid Optical Probes for Neural Membrane Potential Imaging.

The neural membrane potential of nerve cells is the basis of neural activity production, which controls advanced brain activities such as memory, emotion, and learning. In the past decades, optical voltage indicator has emerged as a promising tool to decode neural activities with high-fidelity and excellent spatiotemporal resolution. In particular, the hybrid optical probes can combine the advantageous photophysical properties of different components such as voltage-sensitive molecules, highly fluorescent fluorophores, membrane-targeting tags, and optogenetic materials, thus showing numerous advantages in improving the photoluminescence intensity, voltage sensitivity, photostability, and cell specificity of probes. In this review, the current state-of-the-art hybrid probes are highlighted, that are designed by using fluorescent proteins, organic dyes, and fluorescent nanoprobes as the fluorophores, respectively. Then, the design strategies, voltage-sensing mechanisms and the in vitro and in vivo neural activity imaging applications of the hybrid probes are summarized. Finally, based on the current achievements of voltage imaging studies, the challenges and prospects for design and application of hybrid optical probes in the future are presented.

Anionic Conjugated Polyelectrolytes for FRET-based Imaging of Cellular Membrane Potential.

We report a Förster resonance energy transfer (FRET)-based imaging ensemble for the visualization of membrane potential in living cells. A water-soluble poly(fluorene-cophenylene) conjugated polyelectrolyte (FsPFc10) serves as a FRET donor to a voltage-sensitive dye acceptor (FluoVolt™ ). We observe FRET between FsPFc10 and FluoVolt™ , where the enhancement in FRET-sensitized emission from FluoVolt™ is measured at various donor/acceptor ratios. At a donor/acceptor ratio of 1, the excitation of FluoVolt™ in a FRET configuration results in a three-fold enhancement in its fluorescence emission (compared to when it is excited directly). FsPFc10 efficiently labels the plasma membrane of HEK 293T/17 cells and remains resident with minimal cellular internalization for~1.5h. The successful plasma membrane-associated colabeling of the cells with the FsPFc10-FluoVolt™ donor-acceptor pair is confirmed by dual-channel confocal imaging. Importantly, cells labeled with FsPFc10 show excellent cellular viability with no adverse effect on cell membrane depolarization. During depolarization of membrane potential, HEK 293T/17 cells labeled with the donor-acceptor FRET pair exhibit a greater fluorescence response in FluoVolt™ emission relative to when FluoVolt™ is used as the sole imaging probe. These results demonstrate the conjugated polyelectrolyte to be a new class of membrane labeling fluorophore for use in voltage sensing schemes.

Anti‐proliferative effect of Ca2+‐activated K+ channel KCa2.2 blocker in prostate cancer LNCaP cells

Androgen receptors (AR) are essential for the growth of prostate cancer (PC) cells. Androgen deprivation (castration) therapy as antiandrogens is the powerful treatments for PC. However, the most hormone‐sensitive PC patients after the powerful treatment suffer from progression to hormone‐refractory, castration‐resistant PC (CRPC). Therefore, the novel therapeutic targets are required for PC treatment. Ca2+‐activated K+ (KCa) channels are highly expressed in various types of cancers including prostate cancer, and high level KCa channel expression is associated with high metastatic risk, poor prognosis, and lower overall survival. K+ channel activity was measured by plasma membrane potential imaging with voltage‐sensitive fluorescent dye, DiBAC4(3) and whole‐cell patch clamp recordings, and intracellular Ca2+ concentration was measured by Ca2+ imaging with fluorescent Ca2+ indicator, Fura 2‐AM. Of 5 KCa channels, KCa2.2 was predominantly expressed in human androgen‐sensitive prostate cancer LNCaP cells. The potent and selective KCa2.x blocker, UCL1684‐induced depolarization reduced store‐operated Ca2+ entry (SOCE), and LNCaP cell proliferation was suppressed by the treatment with UCL1684. Both pharmacological and siRNA‐mediated blockades of AR using antiandrogens and AR siRNA decreased the expression levels of KCa2.2 in LNCaP cells, whereas the treatment with UCL1684 did not alter the expression levels of AR, suggesting that KCa2.2 may work as a downstream effector of AR signaling. Of interest, the long‐term androgen deprivation for 96 hr increased the expression levels of AR and KCa2.2 in LNCaP cells. These results provide KCa2.2 may be a potential therapeutic candidate in advanced, recurrent hormone‐sensitive PC and CRPC.

In Vivo Imaging of Mitochondrial Membrane Potential in Non–Small Cell Lung Cancer

In Vivo Imaging of Mitochondrial Membrane Potential in Non–Small Cell Lung Cancer

Nanoparticle-Mediated Visualization and Control of Cellular Membrane Potential: Strategies, Progress, and Remaining Issues.

The interfacing of nanoparticle (NP) materials with cells, tissues, and organisms for a range of applications including imaging, sensing, and drug delivery continues at a rampant pace. An emerging theme in this area is the use of NPs and nanostructured surfaces for the imaging and/or control of cellular membrane potential (MP). Given the important role that MP plays in cellular biology, both in normal physiology and in disease, new materials and methods are continually being developed to probe the activity of electrically excitable cells such as neurons and muscle cells. In this Review, we highlight the current state of the art for both the visualization and control of MP using traditional materials and techniques, discuss the advantageous features of NPs for performing these functions, and present recent examples from the literature of how NP materials have been implemented for the visualization and control of the activity of electrically excitable cells. We conclude with a forward-looking perspective of how we expect to see this field progress in the near term and further into the future.

Publisher Correction: In vivo imaging of mitochondrial membrane potential in non-small-cell lung cancer.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.