MitoTracker Red FM Research Articles

The effect of ventricular microvascular dysfunction on cardiomyocyte excitation-contraction coupling and mitochondria

Abstract Introduction Microvascular dysfunction is a hallmark feature of cardiac allograft vasculopathy in HTx patients and associated with heart failure and coronary artery disease. On a cellular level, impaired mitochondrial respiration has been associated with the disease, however the exact mechanisms remain elusive. Objective The aim of this study was to determine the effect of microvascular dysfunction on functional and structural properties of cardiomyocytes isolated from ventricular biopsies of HTx patients. Methods We functionally assessed coronary artery microvasculature using guidewire based coronary flow reserve(CFR)/microvasculatory resistance (IMR) measurements (Abbott) in 13 post-HTx patients with angiographically normal coronary arteries or normal FFR. Ventricular (RV septum) myocardial biopsies were obtained for in-vitro measurements and single ventricular cardiomyocytes were isolated using enzymatic digestion. Cells were electrically stimulated (1 Hz) and subcellular Ca2+ signalling during excitation-contraction coupling was measured using confocal imaging (Fluo-4 AM). In a subset of cardiomyocytes mitochondrial density was quantified upon staining with Mitotracker Red FM (otsu thresholding). Results Microvascular dysfunction (MVD) measured by IMR was present in 6 of 13 patients with a mean IMR of 56±11 and 13±3 in MVD and CTRL, respectively. CFR did not differ between MVD and CTRL. Ca2+ transients during excitation-contraction coupling in isolated ventricular cardiomyocytes from a subset of patients showed unaltered amplitudes (1.2±0.01 vs. 1.3±0.05; a.u. F/F0). In addition, Ca2+ release (i.e. time to peak Ca2+) and Ca2+ removal (i.e. tau) were not significantly different between MVD and CTRL. However, mitochondrial density was significantly increased in MVD vs. CTRL, indicating subcellular changes associated with MVD. Conclusion In-vivo ventricular microvascular dysfunction is associated with preserved excitation-contraction coupling in-vitro, potentially owing to compensatory changes on the mitochondrial level.

Aggregation-Induced Fluorogens in Bio-Detection, Tumor Imaging, and Therapy: A Review

Aggregation-Induced Fluorogens in Bio-Detection, Tumor Imaging, and Therapy: A Review

Difluoroboron \u03b2-diketonate polylactic acid oxygen nanosensors for intracellular neuronal imaging

Critical for metabolism, oxygen plays an essential role in maintaining the structure and function of neurons. Oxygen sensing is important in common neurological disorders such as strokes, seizures, or neonatal hypoxic–ischemic injuries, which result from an imbalance between metabolic demand and oxygen supply. Phosphorescence quenching by oxygen provides a non-invasive optical method to measure oxygen levels within cells and tissues. Difluoroboron β-diketonates are a family of luminophores with high quantum yields and tunable fluorescence and phosphorescence when embedded in certain rigid matrices such as poly (lactic acid) (PLA). Boron nanoparticles (BNPs) can be fabricated from dye-PLA materials for oxygen mapping in a variety of biological milieu. These dual-emissive nanoparticles have oxygen-insensitive fluorescence, oxygen-sensitive phosphorescence, and rigid matrix all in one, enabling real-time ratiometric oxygen sensing at micron-level spatial and millisecond-level temporal resolution. In this study, BNPs are applied in mouse brain slices to investigate oxygen distributions and neuronal activity. The optical properties and physical stability of BNPs in a biologically relevant buffer were stable. Primary neuronal cultures were labeled by BNPs and the mitochondria membrane probe MitoTracker Red FM. BNPs were taken up by neuronal cell bodies, at dendrites, and at synapses, and the localization of BNPs was consistent with that of MitoTracker Red FM. The brain slices were stained with the BNPs, and the BNPs did not significantly affect the electrophysiological properties of neurons. Oxygen maps were generated in living brain slices where oxygen is found to be mostly consumed by mitochondria near synapses. Finally, the BNPs exhibited excellent response when the conditions varied from normoxic to hypoxic and when the neuronal activity was increased by increasing K+ concentration. This work demonstrates the capability of BNPs as a non-invasive tool in oxygen sensing and could provide fundamental insight into neuronal mechanisms and excitability research.

Evidence for a Physiological Mitochondrial Angiotensin II System in the Kidney Proximal Tubules: Novel Roles of Mitochondrial Ang II/AT1a/O2- and Ang II/AT2/NO Signaling.

The present study tested the hypotheses that overexpression of an intracellular Ang II (angiotensin II) fusion protein, mito-ECFP/Ang II, selectively in the mitochondria of mouse proximal tubule cells induces mitochondrial oxidative and glycolytic responses and elevates blood pressure via the Ang II/AT1a receptor/superoxide/NHE3 (the Na+/H+ exchanger 3)-dependent mechanisms. A PT-selective, mitochondria-targeting adenoviral construct encoding Ad-sglt2-mito-ECFP/Ang II was used to test the hypotheses. The expression of mito-ECFP/Ang II was colocalized primarily with Mito-Tracker Red FM in mouse PT cells or with TMRM in kidney PTs. Mito-ECFP/Ang II markedly increased oxygen consumption rate as an index of mitochondrial oxidative response (69.5%; P<0.01) and extracellular acidification rate as an index of mitochondrial glycolytic response (34%; P<0.01). The mito-ECFP/Ang II-induced oxygen consumption rate and extracellular acidification rate responses were blocked by AT1 blocker losartan (P<0.01) and a mitochondria-targeting superoxide scavenger mito-TEMPO (P<0.01). By contrast, the nonselective NO inhibitor L-NAME alone increased, whereas the mitochondria-targeting expression of AT2 receptors (mito-AT2/GFP) attenuated the effects of mito-ECFP/Ang II (P<0.01). In the kidney, overexpression of mito-ECFP/Ang II in the mitochondria of the PTs increased systolic blood pressure 12±3 mm Hg (P<0.01), and the response was attenuated in PT-specific PT-Agtr1a-/- and PT-Nhe3-/- mice (P<0.01). Conversely, overexpression of AT2 receptors selectively in the mitochondria of the PTs induced natriuretic responses in PT-Agtr1a-/- and PT-Nhe3-/- mice (P<0.01). Taken together, these results provide new evidence for a physiological role of PT mitochondrial Ang II/AT1a/superoxide/NHE3 and Ang II/AT2/NO/NHE3 signaling pathways in maintaining blood pressure homeostasis.

The Dynamic Influence of Olorofim (F901318) on the Cell Morphology and Organization of Living Cells of Aspergillus fumigatus.

The first characterized antifungal in the orotomide class is olorofim. It targets the de novo pyrimidine biosynthesis pathway by inhibiting dihydroorotate dehydrogenase (DHODH). The pyrimidines uracil, thymine and cytosine are the building blocks of DNA and RNA; thus, inhibition of their synthesis is likely to have multiple effects, including affecting cell cycle regulation and protein synthesis. Additionally, uridine-5′-triphosphate (UTP) is required for the formation of uridine-diphosphate glucose (UDP-glucose), which is an important precursor for several cell wall components. In this study, the dynamic effects of olorofim treatment on the morphology and organization of Aspergillus fumigatus hyphae were analyzed microscopically using confocal live-cell imaging. Treatment with olorofim led to increased chitin content in the cell wall, increased septation, enlargement of vacuoles and inhibition of mitosis. Furthermore, vesicle-like structures, which could not be stained or visualized with a range of membrane- or vacuole-selective dyes, were found in treated hyphae. A colocalization study of DHODH and MitoTracker Red FM confirmed for the first time that A. fumigatus DHODH is localized in the mitochondria. Overall, olorofim treatment was found to significantly influence the dynamic structure and organization of A. fumigatus hyphae.

Fluorescent labelling of boar spermatozoa for quantitative studies on competitive sperm-oviduct binding.

Invitro sperm-oviduct binding assays enable assessment of the capacity of spermatozoa to form a 'reservoir' in the oviduct. Competitive approaches, such as experimental set-ups that test multiple males or semen samples simultaneously on the same tissue explants, are desirable because they reduce the likelihood of bias when using material from different females. Therefore, we established a fluorescent labelling technique that allows tagging and storage of spermatozoa before competitive studies of sperm-oviduct binding invitro. Fluorescent markers were tested for reliability and compatibility with parameters of boar spermatozoa viability. The addition of seminal plasma after density gradient centrifugation was essential to counteract centrifugation stress during the labelling procedure. It was demonstrated that sperm tagged with MitoTracker Green FM or MitoTracker Red FM can be successfully used in competitive sperm-oviduct binding studies. The assay was sensitive enough to indicate subtle effects of semen storage temperature on the ability of the spermatozoa to contribute to the female sperm reservoir.

(-)-Epicatechin stimulates mitochondrial biogenesis and cell growth in C2C12 myotubes via the G-protein coupled estrogen receptor

We have reported on the capacity of (-)-epicatechin ((-)-EPI) to stimulate mitochondrial biogenesis (MiB) in mouse skeletal muscle (SkM). However, the mechanisms mediating the effects of (-)-EPI are not fully understood. We previously identified a role of the G-protein coupled estrogen receptor (GPER) in modulating the vascular effects of (-)-EPI. We therefore tested the hypothesis that GPER mediates (at least in part) the stimulatory effects of (-)-EPI on MiB in SkM cells. As an in vitro model, we employed mouse SkM-derived C2C12 myoblasts differentiated into myotubes. Using confocal microscopy, we detected GPER at the cell surface and cytoplasm in C2C12 myotubes. Treatment with (-)-EPI (3 and 10μM) resulted in the stimulation of MiB as per increases in mitochondrial inner (MitoTracker Red FM fluorescence staining) and outer membrane (porin protein levels) markers, transcription factors involved in MiB stimulation (i.e., nuclear respiratory factor-2 [NRF-2] and mitochondrial transcription factor A [TFAM] protein levels) and citrate synthase (CS) activity levels. (-)-EPI-treated myotubes were longer and wider compared to vehicle-treated myotubes. The effects of (-)-EPI on myotube mitochondria and cell size were larger in magnitude to those observed with the GPER agonist G-1. The chemical blockade and down-regulation (siRNA) of GPER evidenced a partial and complete blockade of measured endpoints following (-)-EPI- or G-1-treatment, respectively. Altogether, results indicate that GPER is expressed in muscle cells and appears to mediate to a significant extent, the stimulatory effects of (-)-EPI on MiB. Thus, GPER activation may account for the stimulatory effects of (-)-EPI on SkM structure/function.

Protective Effect of Aspirin Against Oligomeric Aβ42 Induced Mitochondrial Alterations and Neurotoxicity in Differentiated EC P19 Neuronal Cells.

Amyloid-beta (Aβ) induced mitochondrial dysfunction is one of the major causes of neuronal toxicity in Alzheimer's disease. A number of recent reports suggest involvement of mitochondrial alterations through intracellular accumulation of oligomeric Aβ. These mitochondrial alterations include increased Reactive Oxygen Species (ROS), mt-DNA depletion, decreased oxidative phosphorylation and ATP production, membrane depolarization, reduced number of mitochondria etc. All these defects cumulatively caused neural toxicity and alterations in cellular energy homeostasis. On the other hand, anti-inflammatory drug aspirin is reported to promote both mitochondrial biogenesis and improvement in cellular energy status. Taking altogether the mentioned clues, we evaluated protective effect of aspirin, if any on oligomeric Aβ42 induced toxicity and mitochondrial alterations in differentiated neuronal cells. A significant reduction in neuronal viability and increased apoptosis was observed in Aβ42 treated cells, as evident by MTT assay, apoptosis ELISA and immunofluorescence from β-III tubulin antibody staining of neuronal cells. A concomitant decrease was also observed in the intensity of mitotracker red FM staining and mt-DNA to nDNA ratio, suggesting mitochondrial membrane depolarization and/or reduced number of mitochondria along with depletion in mt-DNA. However, simultaneous treatment of 5 μM aspirin to oligomeric Aβ42 treated cells protected them from mitochondrial dysfunction and neurotoxicity. We suggest mitochondrial biogenesis, changes in mitochondrial membrane potential and / or inhibition of Aβ42 aggregation by aspirin as possible underlying mechanism(s).

A mitochondria-targeted colorimetric and two-photon fluorescent probe for biological SO2 derivatives in living cells

Two colorimetric and two-photon fluorescent probes (PCB and MPCB) for SO2 derivatives were designed and synthesized with rapid response, high selectivity, low detection limits, and large Stokes shifts. The probes displayed “turn on” fluorescence response to SO2 derivatives probably due to the nucleophilic addition to the electron-poor CN bond, which was supported by the DFT/TDDFT calculations. Compared with PCB, MPCB was obtained by incorporating a methyl pyridinium group, which endowed MPCB with the red-shifted spectra properties, better water-solubility, larger two-photon excitation action cross-section (δ′) and the important ability of locating in mitochondria. Bio-imaging study established that MPCB can be used to detect SO2 derivatives in living cells under two-photon excitation with large δ′ value (1551 × 0.125 = 193.88 GM), little cytotoxicity and good biocompatibility. Meanwhile, standard co-staining experiments of MPCB and Mito Tracker Red FM (co-localization coefficient: 0.89) revealed that MPCB can be used for the real-time sensing and bioimaging of SO2 derivatives in mitochondria.

The synthesization of BSA-coated iron nanoparticles, and the mechanism of their uptake in the mitochondria of MCF7 breast cancer cells

Event Abstract Back to Event The synthesization of BSA-coated iron nanoparticles, and the mechanism of their uptake in the mitochondria of MCF7 breast cancer cells Mustafa Adham1, 2, Emily Reid1, 2, Devon Scardamaglia1, 2 and Fei Geng1, 3 1 McMaster University, School of Engineering Technology, Canada 2 McMaster University, Student-Biotechnology, School of Engineering Technology, Canada 3 McMaster University, Professor-Biotechnology, School of Engineering Tecnology, Canada Objectives: To test the viability of MCF7 cell lysis through the magnetic properties of iron MNPs which would penetrate the breast cancer cells Verifying iron nanoparticle (MNP) uptake within the mitochondria of the MCF7 cells Materials and Methods: MCF7 cells were grown for 48h then incubated with MNPs for an additional 24h to allow for uptake of the particles into the cells. Iron MNPs are synthesized by a modified Massart coprecipitation method dissolving 1.72g of Iron (II) chloride(FeCl2 4H2O) and 4.67g of Iron (III) chloride(FeCl3 6H2O). The solution was then added to 20 mL ammonium hydroxide(NH4OH) and stirred. The resulting brown precipitate was then centrifuged for 15 min at 5000g and the pellet redispersed in deionized water to remove unreacted reagents and to decrease the pH. After repeating the centrifugation process five times, the MNPs were then pelleted again and redispersed in 20 ml of 5% (w/v) aqueous solution of BSA. In these experiments a batch of MNPs were made with FITC-BSA for imaging. Coating MNPs with BSA caused some aggregation, and sonication was performed for 1 hour using a probe sonicator to separate the MNPs into individual particles. The sonicator pulsed on and off in 5 second intervals to keep BSA from denaturing. The resulting dispersion was a homogeneous liquid when inspected at 400× magnification.The liquid was refrigerated for 24h and then ultracentrifuged at 100,000g for 1 hour. The pellet was redispersed in 40 mL deionized water to remove excess BSA. A 10uL sample of this dispersion was added into the MCF7 Petri-dish, and incubated for another 24h at 37〫 C. The next day, DMEM 1X media was discarded from the dish. The dish was rinsed with PBS, and cells were scraped from the surface, into a centrifuge tube. After 30s centrifugation, the supernatant was discarded. In the experiment with FITC-BSA coated particles, cells were stained according to the procedure for Mito-Tracker ®Red CMXRos FM[5]. In the experiment with BSA coated particles cells were stained according to the staining procedure for Prussian Blue Iron Stain kit[4] Note: H20 replaced with PBS. The same cells were also stained with Mito-Tracker. Results and Discussion: The nanoparticles obtained after sonication were observed with the DLS instrument. The results of the observation showed that the FITC-BSA sample that was sonicated for 1h yielded particles approximately 169.57nm in size. Figure 1 shows the results between three runs where the size distribution is consistent and almost exact. Figure 1 DLS data MCF7 cells incubated with MNPs were observed under an epi-fluorescent microscope to observe MNP uptake within the cell. The image in Figure 2 shows the cells absorbing MNPs coated in FITC-BSA. Figure 2 also shows successful staining of the mitochondria using Mito-Tracker Red FM fluorescent dye. The fluorescent staining seen in the overlay fluorescent image confirms that the nanoparticles are within the mitochondira of the MCF7 cells. Figure 2 FITC-BSA and Mito-Tracker dye Figure 3 Prussian Blue iron stain The results from the epi-fluorescent microscope in Figure 3 confirm the cells successfully absorbing the nanoparticles in the first photo.The cells are stained to induces the Prussian Blue reaction, typically used on tissues to detect the presence of iron.[2] Iron will stain a bright blue andorganelles will stain pink. [3] Image one in Figure 3 clearly shows this phenomenon, proving that iron MNPs have entered the cell. Image two shows the same cell, after being stained with Mito-Tracker. The third image is an overlay, thus proving that FITC-BSA is not what is attracting the iron nanoparticles to the mitochondria. These results prove a novel method in which iron nanoparticles enter the mitochondria of MCF7 breast cancer cells. Paula Gremmen; Dean Ishwar Puri; Dr. Suvojit Ghosh; Eric Mahoney; Dr. James F Hainfeld

Red-Emitting Mitochondrial Probe with Ultrahigh Signal-to-Noise Ratio Enables High-Fidelity Fluorescent Images in Two-Photon Microscopy.

Herein, we reported a red-emitting probe (E)-4-(2-(8-hydroxy-julolidine-9-yl)vinyl)-1-methylpyridin-1-ium iodide (HJVPI) on a rotor mechanism with an ultrahigh signal-to-noise ratio. HJVPI could give high-fidelity fluorescent images of mitochondria in living immortalized and normal cells and be suitable for IR excitation source of two-photon microscopy and various excitation sources of confocal microscopy. As a rotor, its single/two-photon fluorescence intensities directly depended on environmental viscosity. And, as a mitochondrial probe, it displayed much larger two-photon absorption cross sections in comparison with commercial MitoTracker Green FM and MitoTracker Red FM. Moreover, the fact that living cells stained by HJVPI still possessed physiological function could also be confirmed: (1) MTT assay demonstrated that the mitochondria of cells stained retained their electron mediating ability and (2) double assay of HJVPI and SYTOX Blue nucleic acid stain (S-11348) showed that the plasma membrane of the cells stained was still intact. In addition, HJVPI possessed a number of beneficial properties in bioimaging such as good membrane permeability, high photostability, and excellent counterstain compatibility with Hoechst 33342. Related mechanism research suggested that its localization property was dependent on the mitochondrial membrane potential in living cells. All its remarkable properties can extend the investigation on mitochondria in a biological context.

A carbazole-based mitochondria-targeted two-photon fluorescent probe for gold ions and its application in living cell imaging

A carbazole-based two-photon fluorescent probe (PyCM) showed a remarkable fluorescence “turn-on” response to Au3+/Au+ in aqueous solution with a large Stokes shift (119nm). A good linear relationship of emission intensity at 539nm against Au3+/Au+ from 0 to 20 equiv. was observed, and the detection limits (3σ/k) were as low as 47nM for Au3+ and 73nM for Au+, respectively. The fluorescence enhancement was attributed to the gold ions-induced CN bond hydrolysis sensing mechanism, which is fully confirmed by the UV–vis absorption, fluorescence, IR, 1H NMR titration and MALDI-TOF mass analysis. Bio-imaging study established that PyCM could be used to detect Au3+ in living cells under two-photon excitation with large two-photon absorption cross sections (1321 GM at 860nm), little cytotoxicity and good biocompatibility. Meanwhile, standard co-staining experiments of PyCM and MitoTracker Red FM (co-localization coefficient: 0.85) revealed that PyCM was predominantly present in mitochondria.

A mitochondria-targeted two-photon fluorescent probe for highly selective and rapid detection of hypochlorite and its bio-imaging in living cells

Two water-soluble carbazole-based two-photon fluorescent (TPF) probes (HCH and HCM) for ClO− detection were developed with rapid response, high selectivity and large two-photon absorption cross sections, which utilized the oxidative dehydrogenation of oxime to the nitrile oxide. It is found that the alkyl substituent groups in the cationic pyridinium moieties could affect the one-/two-photon luminescent properties of probes, and HCH was more suitable for ClO− detection than HCM thanks to the better stability of fluorescence, larger fluorescence enhancement folds and two-photon absorption cross sections for HCHCNO, the corresponding product of HCH toward ClO−. Bio-imaging study revealed that HCH could be used to detect ClO− in living cells under two-photon excitation, and co-staining experiments of HCH and MitoTracker Red FM (co-localization coefficient: 0.90) established that HCH was predominantly present in mitochondria.

Apelin Enhances Brown Adipogenesis and Browning of White Adipocytes

Brown adipose tissue expends energy in the form of heat via the mitochondrial uncoupling protein UCP1. Recent studies showed that brown adipose tissue is present in adult humans and may be exploited for its anti-obesity and anti-diabetes actions. Apelin is an adipocyte-derived hormone that plays important roles in energy metabolism. Here, we report that apelin-APJ signaling promotes brown adipocyte differentiation by increasing the expressions of brown adipogenic and thermogenic transcriptional factors via the PI3K/Akt and AMPK signaling pathways. It is also found that apelin relieves the TNFα inhibition on brown adipogenesis. In addition, apelin increases the basal activity of brown adipocytes, as evidenced by the increased PGC1α and UCP1 expressions, mitochondrial biogenesis, and oxygen consumption. Finally, we provide both in vitro and in vivo evidence that apelin is able to increase the brown-like characteristics in white adipocytes. This study, for the first time, reveals the brown adipogenic and browning effects of apelin and suggests a potential therapeutic route to combat obesity and related metabolic disorders.

Visualization and enrichment of live putative cancer stem cell populations following p53 inactivation or Bax deletion using non-toxic fluorescent dyes

Putative cancer stem cell (CSC) populations efflux dyes such as Hoecsht 33342 giving rise to side-populations (SP) that can be analyzed or isolated by flow cytometry. However, Hoecsht 33342 is highly toxic, more so to non-SP cells, and thus presents difficulties in interpreting in vivo studies where non-SP cells appear less tumorigenic than SP cells in immunodeficient mice. We searched for non- toxic dyes to circumvent this problem as well as to image these putative CSCs. We found that the fluorescent dye calcein, a product of intracellular Calcein AM cleavage, is effluxed by a small subpopulation, calcein low population (CloP). This population overlaps with SP and demonstrated long term cell viability, lack of cell stress, and proliferation in several cancer cell lines when stained whereas Hoecsht 33342 staining caused substantial apoptosis and ablated proliferation. We also found that the effluxed dye D-luciferin exhibits strong UV-fluorescence that can be imaged at cellular resolution and spatially overlaps with Calcein AM. In order to evaluate the hypothesis that p53 loss promotes enrichment of putative CSC populations we used Calcein AM, D-luciferin, and Mitotracker Red FM as a counterstain to visualize dye-effluxing cells. Using fluorescence microscopy and flow cytometry we observed increased dye-effluxing populations in DLD-1 colon tumor cells with mutant p53 versus wild-type (WT) p53-expressing HCT116 cells. Deletion of the wild-type p53 or pro-apoptotic Bax genes induced the putative CSC populations in the HCT116 background to significant levels. Restoration of WT p53 in HCT116 p53-/- cells by an adenovirus vector eliminated the putative CSC populations whereas a control adenovirus vector, Ad-LacZ, maintained the putative CSC population. Our results suggest it is possible to image and quantitatively analyze putative CSC populations within the tumor microenvironment and that loss of pro-apoptotic and tumor suppressing genes such as Bax or p53 enrich such tumor-prone populations.