Tetramethylrhodamine Methyl Ester Research Articles

A new fluorescent dye for cell tracing and mitochondrial imaging in vitro and in vivo.

Mitochondria contribute to redox and calcium balance, and apoptosis thus regulating cellular fate. In the present study, mitochondrial staining applying a novel dye, V07-07059, was performed in human embryonic kidney cells, a human vascular endothelial cell line and primary human mononuclear cells. The new fluorescent mega Stokes dye (peak excitation: 488nm, peak emission: 554nm) showed superior fluorescent properties and stability. V07-07059 stains mitochondria dependent on their membrane potential and is safe to use in vitro and in vivo. Unlike other dyes applied in this context (e.g. Tetramethylrhodamine methyl ester), V07-07059 only marginally inhibits mitochondrial respiration and function. V07-07059 enables real time imaging of mitochondrial trafficking and remodeling. Prolonged staining with V07-07059 demonstrated the dyes suitability as a novel probe to track cells. In comparison to the widely used standard for cell proliferation and tracking studies 5(6)-diacetate N-succinimidyl ester, V07-07059 proved superior regarding toxicity and photostability.

PKA Phosphorylation of NCLX Reverses Mitochondrial Calcium Overload and Depolarization, Promoting Survival of PINK1-Deficient Dopaminergic Neurons

SUMMARYMitochondrial Ca2+ overload is a critical, preceding event in neuronal damage encountered during neurodegenerative and ischemic insults. We found that loss of PTEN-induced putative kinase 1 (PINK1) function, implicated in Parkinson disease, inhibits the mitochondrial Na+/Ca2+ exchanger (NCLX), leading to impaired mitochondrial Ca2+ extrusion. NCLX activity was, however, fully rescued by activation of the protein kinase A (PKA) pathway. We further show that PKA rescues NCLX activity by phosphorylating serine 258, a putative regulatory NCLX site. Remarkably, a constitutively active phosphomimetic mutant of NCLX (NCLXS258D) prevents mitochondrial Ca2+ overload and mitochondrial depolarization in PINK1 knockout neurons, thereby enhancing neuronal survival. Our results identify an mitochondrial Ca2+ transport regulatory pathway that protects against mitochondrial Ca2+ overload. Because mitochondrial Ca2+ dyshomeostasis is a prominent feature of multiple disorders, the link between NCLX and PKA may offer a therapeutic target.

Abstract 077: In Vivo Evidence of AT1a Receptor-Mediated Uptake of Angiotensin II by the Proximal Tubule Visualized by Intravital Multiphoton Imaging

The development of all forms of angiotensin II (ANG II)-dependent hypertension is associated with higher levels of intrarenal ANG II, which are greater than can be explained on the basis of circulating ANG II and suppressed cortical renin expression. In the present study, we used intravital multiphoton imaging to test the hypothesis that AT1 (AT1a) receptor-mediated uptake of ANG II by the proximal tubules of the kidney plays a major role in the underlying mechanisms. Adult male Munich-Wistar rats were anesthetized with Inactin and continuously infused with a pressor dose of Alexa 488-conjugated ANG II (50 ng/min, i.v.) for 2 hr. Time-dependent proximal tubular uptake responses of Alexa 488-ANG II were studied with mean arterial blood pressure maintained at ~150 mmHg throughout the experiment. After 30 min infusion, very low levels of Alexa 488-ANG II were visualized within proximal tubules and cortical collecting ducts (CCDs) (p<0.05). However, high levels of Alexa 488-ANG II were accumulated in the lumen of CCDs, but not that of proximal tubules. After 1 hr infusion, moderate levels of Alexa 488-ANG II were visualized in the proximal tubules, but not in the glomeruli and CCDs. The most striking uptake responses were visualized in all segments of proximal tubules after 2 hr infusion. Internalized Alexa 488-ANG II was predominantly localized to the basolateral side, where it was colocalized with tetramethyl rhodamine methyl ester (TMRM), a mitochondrial membrane potential-dependent dye. TMRM is a lipophilic cationic dye that is primarily accumulated in the mitochondria of proximal tubules. Some internalized Alexa 488-ANG II was visualized around and over the nuclei. Furthermore, the uptake of Alexa 488-ANG II by proximal tubules was significantly attenuated in caveolin 1-knockout mice (p<0.01), and blocked in AT1a receptor-knockout mice (p<0.01). Our results provide strong intravital multiphoton microscopic evidence that circulating ANG II is primarily taken up by the proximal tubules of the kidney via an AT1a receptor-mediated mechanism, and that internalized ANG II may be transported to the mitochondria and the nucleus, where it may alter mitochondrial and nuclear function in the proximal tubules of the kidney.

Detection of Mitochondrial Mass, Damage, and Reactive Oxygen Species by Flow Cytometry.

The reagents and procedures highlighted here will give the investigators an indication of the health status and volume of mitochondria in primary cells and cell lines through the use of a number of cell-permeable dyes. Mitochondrial volume can be monitored by using the probe MitoTracker Green FM. This reagent labels mitochondria in a manner that is independent of the membrane potential, therefore providing a readout relating purely to the mitochondrial mass of the cell. In contrast, MitoTracker Red CMXRos, tetramethylrhodamine methyl ester, and 10-N-nonyl acridine orange label mitochondria in a manner dependent on the membrane potential, thus giving an indication of mitochondrial stress. Using MitoSOX Red, it is also possible to analyze the production of the mitochondrial superoxide anion. Like the MitoTracker probes, MitoSOX Red is taken up passively by cells. In the mitochondria, the probe is oxidized by superoxide, resulting in the emission of red fluorescence.

Abstract 364: Pioglitazone Inhibition of Vascular Smooth Muscle Cell Proliferation is Associated with Activation of AMP-activated Protein Kinase but not Peroxisome Proliferator-Activated Receptor Gamma

Atherosclerosis and restenosis after angioplasty are associated with exaggerated proliferation of vascular smooth muscle cells (VSMCs). Pioglitazone (PIO), an antidiabetic drug that belongs to the family of peroxisome proliferator-activated receptor gamma (PPARg) agonists, reduces intimal hyperplasia after coronary angioplasty in diabetic and nondiabetic subjects. However, the molecular mechanisms by which PIO regulates VSMC phenotype remain unclear. The objective of the current study is to determine the role of AMP-activated protein kinase (AMPK, a cellular energy sensor) versus PPARg in PIO-induced changes in VSMC phenotype. Exposure of human aortic VSMCs to PIO followed by immunoblot analysis revealed a robust increase in the phosphorylation of AMPK (Thr172) and its downstream effector, Acetyl-CoA carboxylase (p < 0.05, n = 3), as a function of concentration (3 to 30 μM) and time (1 to 48 hr). Notably, PIO (30 μM, 48 hr) activation of AMPK remained essentially the same after PPARg downregulation (~90%) by target-specific siRNA (500 pmoles), suggesting lack of dependency on PPARg. In addition, PIO activation of AMPK was accompanied by induction of mitochondrial membrane depolarization with a trend toward an increase in AMP/ATP ratio (~40%), as revealed by tetramethylrhodamine methyl ester (TMRM) fluorescent redistribution & LC-MS/MS MRM analysis, respectively. Importantly, PIO pretreatment (30 μM, 30 min) resulted in significant inhibition of PDGF (30 ng/ml, 48 hr)-induced VSMC proliferation by ~85% (cell count) and the associated increases in proliferative markers (cyclin D1 and pRb) by > 95% (p < 0.05, n = 3). Downregulation of AMPKa1 (~80%) by target-specific AMPKa1 siRNA (500 pmoles) revealed that PIO-mediated AMPK activation inhibited mTORC1 signaling by > 95% & upregulated cell cycle inhibitor p27Kip1 by ~40%, thus accounting for PIO-mediated changes in VSMC phenotype. In vivo, PIO administration (10 mg/Kg/d, oral gavage) for 3 weeks in CJ57BL6 mice mitigated neointimal growth by ~60% in guide wire-injured femoral artery (p < 0.05, n = 8). In conclusion, the present study provides the first direct evidence for VSMC-specific AMPK activation by PIO, which may in part contribute to its vasoprotective effects in nondiabetic state.

Saffron extracts alleviate cardiomyocytes injury induced by doxorubicin and ischemia-reperfusion in vitro

Doxorubicin (DOX), a highly active chemotherapeutic drug, faces limitations in clinical application due to severe cardiotoxic effects (mainly through increased oxidative stress). Therefore, its effect is exacerbated in subjects with ischemic heart disease. We have recently reported that saffron extract (SAF), a natural compound mainly consisting of safranal and corcins, exerts a protective effect against DOX oxidative cytotoxicity in isolated rabbit hearts. Here, we aimed to investigate whether SAF exerts cardioprotection against combined ischemia-reperfusion (I/R) and DOX toxicity in H9c2 cardiomyocytes. H9c2 were subjected to simulated I/R, with or without DOX treatment at reperfusion, in the presence or absence of SAF prior to ischemia or at reperfusion. We evaluated the effects of these treatments by MTT, LDH and western blot analysis. Apoptosis was assessed by Hoechst 33258 staining, tetramethyl rhodamine methyl ester fluorescence and caspase activity. The results showed that I/R and DOX significantly decreased cardiomyocytes viability, inhibited reperfusion injury salvage kinase cardioprotective pathway, reduced contractile proteins (α-Actinine, Troponine C and MLC), increased caspase-3 expression and induced loss of mitochondrial membrane potential. These effects were remarkably inhibited by treatment with SAF (10 μg/mL) at reperfusion. SAF activated AKT/P70S6K and ERK1/2, restored contractile proteins expression, inhibited mitochondrial permeability transition pore and decreased caspase-3 activity. In conclusion, our findings indicate that SAF treatment exerted cardioprotection against I/R and DOX toxicity by reducing oxidative stress (LDH assay). Thereby, SAF offers a potential novel antioxidant therapeutic strategy to counteract I/R and DOX cardiotoxicity, paving the way for future clinical trials.

VDAC Opening Drugs to Induce Mitochondrial Dysfunction and Cell Death

Background: Mitochondrial membrane potential (ΔΨ) and reactive oxygen species (ROS) formation depend on metabolite flux into mitochondria through voltage dependent anion channels (VDAC). Free tubulin closes VDAC, and high free tubulin levels decrease ΔΨ in cancer cells. Erastin opens VDAC by antagonizing the inhibitory effect of tubulin. Here, we hypothesized that erastin and erastin-like compounds open VDAC, increase mitochondrial metabolism and ROS formation, and activate c-jun N-terminal kinase (JNK), leading to mitochondrial dysfunction and cell killing. Our AIM was to evaluate the effects of erastin/erastin-like compounds on ΔΨ, ROS, NADH, JNK activation, cell killing and protection by antioxidants in HepG2 cells.Methods: Confocal/multiphoton fluorescence microscopy assessed ΔΨ (tetramethylrhodamine methylester), ROS (chloromethyldichlorofluorescein [cmDCF], MitoSOX Red) and NADH (autofluorescence). JNK was assessed by Western blotting and cell killing by propidium iodide assay.Results: Erastin increased ΔΨ by 46% and NADH by 30% and blocked the depolarizing effect of microtubule destabilizers in HepG2 human hepatoma cells. Increased ΔΨ after erastin/erastin-like compounds induced mitochondrial hyperpolarization that was followed by depolarization. Small molecules X1 and X2, identified in a high-throughput screening, caused a more rapid drop of ΔΨ (<1 h) compared to erastin (3-4 h). Erastin, X1 and X2 also maximally increased cmDCF and MitoSOX Red fluorescence after 1-2 h. Additionally, JNK activation peaked at 60 min. JNK activation and ROS formation preceded mitochondrial depolarization. Cell killing promoted by X1 (93%) and X2 (76%) after 12 h was blocked by the antioxidant N-acetyl cysteine (100 µM).Conclusion: Mitochondrial hyperpolarization caused by VDAC opening drugs causes oxidative stress, which in turn leads to JNK activation, mitochondrial dysfunction and cell death that is prevented by antioxidants. Grants DK073336, DK037034 and 14.Z50.31.0028 to JJL and ACS 13-043-01 to ENM.

Mitochondrial depolarization and asystole in the globally ischemic rabbit heart: coordinated response to interventions affecting energy balance.

Mitochondrial membrane potential (ΔΨm) depolarization has been implicated in the loss of excitability (asystole) during global ischemia, which is relevant for the success of defibrillation and resuscitation after cardiac arrest. However, the relationship between ΔΨm depolarization and asystole during no-flow ischemia remains unknown. We applied spatial Fourier analysis to confocally recorded fluorescence emitted by ΔΨm-sensitive dye tetramethylrhodamine methyl ester. The time of ischemic ΔΨm depolarization (tmito_depol) was defined as the time of 50% decrease in the magnitude of spectral peaks reflecting ΔΨm. The time of asystole (tasys) was determined as the time when spontaneous and induced ventricular activity ceased to exist. Interventions included tachypacing (150 ms), myosin II ATPase inhibitor blebbistatin (heart immobilizer), and the combination of blebbistatin and the inhibitor of glycolysis iodoacetate. In the absence of blebbistatin, confocal images were obtained during brief perfusion with hyperkalemic solution and after the contraction failed between 7 and 15 min of ischemia. In control, tmito_depol and tasys were 24.4 ± 6.0 and 26.0 ± 5.0 min, respectively. Tachypacing did not significantly affect either parameter. Blebbistatin dramatically delayed tmito_depol and tasys (51.4 ± 8.6 and 45.7 ± 5.3 min, respectively; both P < 0.0001 vs. control). Iodoacetate combined with blebbistatin accelerated both events (tmito_depol, 12.7 ± 1.8 min; and tasys, 6.5 ± 1.1 min; both P < 0.03 vs. control). In all groups pooled together, tasys was strongly correlated with tmito_depol (R(2) = 0.845; P < 0.0001). These data may indicate a causal relationship between ΔΨm depolarization and asystole or a similar dependence of the two events on energy depletion during ischemia. Our results urge caution against the use of blebbistatin in studies addressing pathophysiology of myocardial ischemia.

Quantitative analysis of mitochondrial morphology and membrane potential in living cells using high-content imaging, machine learning, and morphological binning

Understanding the processes of mitochondrial dynamics (fission, fusion, biogenesis, and mitophagy) has been hampered by the lack of automated, deterministic methods to measure mitochondrial morphology from microscopic images. A method to quantify mitochondrial morphology and function is presented here using a commercially available automated high-content wide-field fluorescent microscopy platform and R programming-language-based semi-automated data analysis to achieve high throughput morphological categorization (puncta, rod, network, and large & round) and quantification of mitochondrial membrane potential. In conjunction with cellular respirometry to measure mitochondrial respiratory capacity, this method detected that increasing concentrations of toxicants known to directly or indirectly affect mitochondria (t-butyl hydroperoxide [TBHP], rotenone, antimycin A, oligomycin, ouabain, and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone [FCCP]), decreased mitochondrial networked areas in cultured 661w cells to 0.60–0.80 at concentrations that inhibited respiratory capacity to 0.20–0.70 (fold change compared to vehicle). Concomitantly, mitochondrial swelling was increased from 1.4- to 2.3-fold of vehicle as indicated by changes in large & round areas in response to TBHP, oligomycin, or ouabain. Finally, the automated identification of mitochondrial location enabled accurate quantification of mitochondrial membrane potential by measuring intramitochondrial tetramethylrhodamine methyl ester (TMRM) fluorescence intensity. Administration of FCCP depolarized and administration of oligomycin hyperpolarized mitochondria, as evidenced by changes in intramitochondrial TMRM fluorescence intensities to 0.33- or 5.25-fold of vehicle control values, respectively. In summary, this high-content imaging method accurately quantified mitochondrial morphology and membrane potential in hundreds of thousands of cells on a per-cell basis, with sufficient throughput for pharmacological or toxicological evaluation.

Effects of 915 nm GaAs diode laser on mitochondria of human dermal fibroblasts: analysis with confocal microscopy.

Low-level laser therapy (LLLT) is widely used in tissue regeneration and pain therapy. Mitochondria are supposed to be one of the main cellular targets, due to the presence of cytochrome C oxidase as photo-acceptor. Laser stimulation could influence mitochondria metabolism affecting mainly transmembrane mitochondrial potential (Δψm). The aim of our study is to evaluate "in vitro" the early mitochondrial response after irradiation with a 915 GaAs laser. Since some evidences suggest that cellular response to LLLT can be differently modulated by the mode of irradiation, we would like to evaluate whether there are changes in the mitochondrial potential linked to the use of the laser treatments applied with continuous wave (CW) in respect to those applied with pulsed wave (PW). In this study, we analyzed effects of irradiation with a 915-nm GaAs diode laser on human dermal fibroblast. We compared effects of irradiation applied with either CW or PW at different fluences 45-15-5 J/cm(2) on Δψm. Laser scanning microscopy (LSM) was used in living cells to detect ROS (reactive oxygen species) using calcein AM and real-time changes of and Δψm following distribution of the potentiometric probe tetramethylrhodamine methyl ester (TMRM). At higher doses (45-15 J/cm(2)), fibroblasts showed a dose-dependent decrement of Δψm in either the modalities employed, with higher amplitudes in CW-treated cells. This behavior is transient and not followed by any sign of toxicity, even if reactive oxygen species generation was observed. At 5 J/cm(2), CW irradiation determined a little decrease (5%) of the baseline level of Δψm, while opposite behavior was shown when cells were irradiated with PW, with a 10% increment. Our results suggest that different responses observed at cellular level with low doses of irradiation, could be at the basis of efficacy of LLLT in clinical application, performed with PW rather than CW modalities.

Water-soluble coenzyme q10 inhibits nuclear translocation of apoptosis inducing factor and cell death caused by mitochondrial complex I inhibition.

The objectives of the study were to explore the mechanism of rotenone-induced cell damage and to examine the protective effects of water-soluble Coenzyme Q10 (CoQ10) on the toxic effects of rotenone. Murine hippocampal HT22 cells were cultured with mitochondrial complex I inhibitor rotenone. Water-soluble CoQ10 was added to the culture media 3 h prior to the rotenone incubation. Cell viability was determined by alamar blue, reactive oxygen species (ROS) production by dihydroethidine (DHE) and mitochondrial membrane potential by tetramethyl rhodamine methyl ester (TMRM). Cytochrome c, caspase-9 and apoptosis-inducing factor (AIF) were measured using Western blotting after 24 h rotenone incubation. Rotenone caused more than 50% of cell death, increased ROS production, AIF nuclear translocation and reduction in mitochondrial membrane potential, but failed to cause mitochondrial cytochrome c release and caspase-9 activation. Pretreatment with water-soluble CoQ10 enhanced cell viability, decreased ROS production, maintained mitochondrial membrane potential and prevented AIF nuclear translocation. The results suggest that rotenone activates a mitochondria-initiated, caspase-independent cell death pathway. Water-soluble CoQ10 reduces ROS accumulation, prevents the fall of mitochondrial membrane potential, and inhibits AIF translocation and subsequent cell death.

Abstract 121: Combinatorial Tailored Polymers Enhanced Maturation of Human iPSC-CMs

There is a tremendous interest in human cardiomyocytes generated from patient-derived induced pluripotent stem cells (iPSC-CMs) for the study and possible treatment of human heart diseases. Despite their vast potential, a significant impediment to a broader application of iPSC-CMs to study human myocyte biology is the structural and functional immaturity of iPSC-CMs. Growing evidence indicates that synthetic polymers utilized as extracellular substrates can exert significant effects on in vitro tissue generation, although the underlying mechanisms remain largely unknown. Based on the profound impact of the extracellular matrix of developing embryos on in vivo organogenesis, we hypothesize that engineered polymer substrates will likewise influence in vitro maturation of iPSC-CMs. A subset of combinatorial polymers was synthesized by polymerizing poly(ε-caprolacton) (PCL), polyethylene glycol (PEG), and carboxylated PCL (cPCL), abbreviated as x%PEG-y%PCL-z%cPCL (x, y, and z: molar %). We investigated effects of the polymer composition on maturation of iPSC-CMs with respect to the beating behavior, mitochondrial function and molecular profiles after 30 days in culture on polymer scaffolds. Results showed the 4%PEG-96%PCL scaffold promoted the most active beating in iPSC-CMs at 30 days and further, that the mitochondrial function, as assessed by tetramethyl rhodamine methylester (TMRM) was significantly increased in the iPSC-CMs cultured on 4%PEG-96%PCL over other polymers. Molecular profiling analysis indicates 4%PEG-96%PCL scaffolds enhanced the expression of MYL2 (a commonly accepted marker of mature ventricular myocytes) as well as of components of the intermediate filaments linking the plasma membrane to the myofilament. In summary, although the polymers we used here exhibit similar physicochemical properties, they have divergent effects on iPSC-CM differentiation. Thus, specific chemical compositions of synthetic substrates can exert profound influence on in vitro maturation of hiPSC-CMs. Our work exploring the effects of synthetic biomaterials on human stem cell differentiation could pave the way for a successful translation of ongoing advances in tissue engineering to new treatments for human heart diseases.

Physiological and molecular level effects of silver nanoparticles exposure in rice (Oryza sativa L.) seedlings

The physiological and molecular level changes of silver nanoparticles (AgNPs) exposure were investigated in rice (Oryza sativa L.) seedlings. The seedlings were exposed to different concentrations of (0, 0.2, 0.5 and 1mgL−1) AgNPs for one week. Significant reduction in root elongation, shoot and root fresh weights, total chlorophyll and carotenoids contents were observed. Exposure to 0.5 and 1mgL−1 of AgNPs caused significant increase in hydrogen peroxide formation and lipid peroxidation in shoots and roots, increased foliar proline accumulation and decreased sugar contents. AgNPs exposure resulted in a dose dependant increase in reactive oxygen species generation and also caused cytotoxicity as evidenced by increased dihydroethidium, 3′-(p-hydroxyphenyl) fluorescein and propidium iodide fluorescence. Tetramethylrhodamine methyl ester assay showed decreased mitochondrial membrane potential with increasing concentrations of AgNPs exposure in roots. Real Time PCR analysis showed differential transcription of genes related to oxidative stress tolerance viz. FSD1, MSD1, CSD1, CSD2, CATa, CATb, CATc, APXa and APXb in shoots and roots of rice seedlings. The overall results suggest that exposure to AgNPs caused significant physiological and molecular level changes, oxidative stress and also resulted in the induction oxidative stress tolerance mechanisms in rice seedlings.

TRAP1 Regulates Proliferation, Mitochondrial Function, and Has Prognostic Significance in NSCLC

The TNF receptor-associated protein 1 (TRAP1) is a mitochondrial HSP that has been related to drug resistance and protection from apoptosis in colorectal and prostate cancer. Here, the effect of TRAP1 ablation on cell proliferation, survival, apoptosis, and mitochondrial function was determined in non-small cell lung cancer (NSCLC). In addition, the prognostic value of TRAP1 was evaluated in patients with NSCLC. These results demonstrate that TRAP1 knockdown reduces cell growth and clonogenic cell survival. Moreover, TRAP1 downregulation impairs mitochondrial functions such as ATP production and mitochondrial membrane potential as measured by TMRM (tetramethylrhodamine methylester) uptake, but it does not affect mitochondrial density or mitochondrial morphology. The effect of TRAP1 silencing on apoptosis, analyzed by flow cytometry and immunoblot expression (cleaved PARP, caspase-9, and caspase-3) was cell line and context dependent. Finally, the prognostic potential of TRAP1 expression in NSCLC was ascertained via immunohistochemical analysis which revealed that high TRAP1 expression was associated with increased risk of disease recurrence (univariate analysis, P = 0.008; multivariate analysis, HR: 2.554; 95% confidence interval, 1.085-6.012; P = 0.03). In conclusion, these results demonstrate that TRAP1 impacts the viability of NSCLC cells, and that its expression is prognostic in NSCLC. TRAP1 controls NSCLC proliferation, apoptosis, and mitochondrial function, and its status has prognostic potential in NSCLC.

Role of mitochondrial KATP channels in PKCβ‐dependent constriction of small pulmonary arteries (1090.10)

PKCβ mediates vasoconstriction through a unique signaling pathway in the pulmonary circulation that involves mitochondrial reactive oxygen species (mitoROS). We hypothesized that PKCβ elicits these responses through activation of vascular smooth muscle mitochondrial KATP channels (mitoKATP). To test this hypothesis, we examined constriction in endothelium‐disrupted, pressurized rat pulmonary arteries (~150 μm diameter) and mitoROS production (by MitoSOX fluorescence) in cultured rat and human pulmonary arterial smooth muscle cells (PASMC) in response to the PKC agonist phorbol 12‐myristate 13‐acetate (PMA). PMA caused vasoconstriction and mitoROS production that were significantly attenuated (p&lt;0.05) by both the PKCβ inhibitor LY‐333,531 (10 nM) and PKCβ siRNA. Consistent with our hypothesis, the specific mitoKATP channel antagonist 5‐hydroxydecanoate (5‐HD; 500 μM) inhibited vasoconstriction and mitoROS production to both PMA and the mitoKATP activator diazoxide (10‐5‐ 10‐3 M). Consistent with a role for mitoKATP in PKCβ‐dependent mitoROS generation, PMA caused mitochondrial membrane potential depolarization (detected by tetramethylrhodamine methyl ester fluorescence), which was prevented by 5‐HD (p&lt;0.05 vs. vehicle) in both pulmonary arteries and human PASMC. We conclude that PKCβ enhances mitoROS production via mitoKATP activation in PASMC to mediate vasoconstriction.Grant Funding Source: Supported by NIH R01 HL088192 and American Heart Association grants 1POST5260026 and 13GRNT14

High-Content Imaging Assays for Identifying Compounds that Generate Superoxide and Impair Mitochondrial Membrane Potential in Adherent Eukaryotic Cells.

Reactive oxygen species (ROS) are constantly produced in cells as a result of aerobic metabolism. When there is an excessive production of ROS and the cell's antioxidant defenses are overwhelmed, oxidative stress occurs. The superoxide anion is a type of ROS that is produced primarily in mitochondria but is also generated in other regions of the cell including peroxisomes, endoplasmic reticulum, plasma membrane, and cytosol. Here, a high-content imaging assay using the dye dihydroethidium is described for identifying compounds that generate superoxide in eukaryotic cells. A high-content imaging assay using the fluorescent dye tetramethylrhodamine methyl ester is also described to identify compounds that impair mitochondrial membrane potential in eukaryotic cells. The purpose of performing both assays is to identify compounds that (1) generate superoxide at lower concentrations than they impair mitochondrial membrane potential, (2) impair mitochondrial membrane potential at lower concentrations than they generate superoxide, (3) generate superoxide and impair mitochondrial function at similar concentrations, and (4) do not generate superoxide or impair mitochondrial membrane potential during the duration of the assays.

Ipratropium Bromide-Mediated Myocardial Injury in In Vitro Models of Myocardial Ischaemia/Reperfusion

Ipratropium bromide, a nonselective muscarinic antagonist, is widely prescribed for the treatment of chronic obstructive pulmonary disease (COPD). Analyses of COPD patients, with underlying ischaemic heart disease, receiving anticholinergics, have indicated increased risk of severity and occurrence of cardiovascular events (including myocardial infarction). The present study explored whether ipratropium bromide induces myocardial injury in nonclinical models of simulated myocardial ischaemia/reperfusion injury. Adult Sprague Dawley rat hearts/primary ventricular myocytes were exposed to simulated ischaemia/hypoxia prior to administration of ipratropium at the onset of reperfusion/reoxygenation. Infarct to risk ratio and cell viability was measured via triphenyl tetrazolium chloride staining and thiazolyl blue tetrazolium bromide (MTT) assay. The involvement of apoptosis and necrosis was evaluated by flow cytometry. Mitochondrial-associated responses were detected by tetramethylrhodamine methyl ester fluorescence and myocyte contracture. Ipratropium (1 × 10⁻¹¹ M - 1 × 10⁻⁴ M) significantly increased infarct/risk ratio and decreased cell viability in a dose-dependent manner. Increased levels of necrosis and apoptosis were observed via flow cytometry, accompanied by increased levels of cleaved caspase-3 following ipratropium treatment. Levels of endogenous myocardial acetylcholine were verified via use of an acetylcholine assay. In these experimental models, exogenous acetylcholine (1 × 10⁻⁷ M) showed protective properties, when administered alone, as well as abrogating the exacerbation of myocardial injury during ischaemia/reperfusion following ipratropium coadministration. In parallel experiments, under conditions of myocardial ischaemia/reperfusion, a similar injury was observed following atropine (1 × 10⁻⁷ M) administration. These data demonstrate for the first time in a nonclinical setting that ipratropium exacerbates ischaemia/reperfusion injury via apoptotic- and necrotic-associated pathways.

Antagonists of the Inhibitory Effect of Free Tubulin on VDAC Induce Oxidative Stress and Mitochondrial Dysfunction

Bakground: Mitochondrial membrane potential (ΔΨ) and generation of reactive oxygen species (ROS) requires a respiratory chain fueled by the flux of metabolites into mitochondria through voltage dependent anion channels (VDAC). Free tubulin induces reversible blockage of VDAC both in vitro and in cells. Erastin, a small molecule lethal to cancer cells, antagonizes the inhibitory effect of free tubulin on VDAC and upregulates mitochondrial metabolism. Here, we hypothesized that erastin and erastin-like compounds open VDAC, increase mitochondrial metabolism and ROS formation, and activate JNK, which in turn cause mitochondrial dysfunction. Our AIM was to evaluate the effects of erastin/erastin-like compounds on ΔΨ, NADH, ROS and JNK in intact cells. METHODS: ΔΨ was assessed by confocal microscopy of tetramethylrhodamine methylester (TMRM) fluorescence and ROS by chloromethyldichlorofluorescein (cmDCF) and MitoSOX Red fluorescence. Mitochondrial NADH autofluorescence was assessed by multiphoton microscopy. Total and phosphorylated JNK was determined by Western blotting. RESULTS: In lipid bilayers, erastin antagonized tubulin inhibition on VDAC. In HepG2 human hepatoma cells, erastin increased ΔΨ by 46% and NADH by 30%. Mitochondrial hyperpolarization plateaued after 2 h. Subsequently depolarization occurred (3-4 h), suggesting mitochondrial dysfunction. Erastin-like compounds X1 and X2, identified in a high-throughput screening, similarly caused mitochondrial hyperpolarization/depolarization. Erastin also caused increases of DCF and Mitosox Red fluorescence beginning after ∼30 min and reaching a maximum after 2 h. Additionally, erastin activated JNK (maximum pJNK at 60 min). JNK activation and ROS formation both preceded mitochondrial depolarization. Conclusion: Erastin relieves tubulin-dependent inhibition of VDAC conductance, leading to mitochondrial hyperpolarization that increases ROS production with concomitant activation of the stress kinase JNK. These events, in turn, may induce the mitochondrial permeability transition, mitochondrial dysfunction and ultimately cell death.

Targeted suppression of glutaryl-CoA dehydrogenase by lentivirus-mediated SHRNA and excessive intake of lysine induce the apoptosis of rat striatal neurons

Methods Four short hairpin RNA (shRNA) sequences targeting the GCDH gene (NM_001108896) were designed to construct four recombinant lentiviral vectors. The effectiveness of gene silencing in rat striatal neurons was detected by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting techniques. GCDH deficiency neurons (GCDH neurons), neurons transfected with negative control virus (NC neurons) and not intervention neurons (C neurons) were all incubated with lysine for 24h in concentrations of 0mmol/L, 5mmol/L, 10mmol/L respectively. The viability was measured with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT). The apoptosis of the neurons were detected by Hoechst33342 and PI. Tetramethylrhodamine methyl ester (TMRM) was used to determine the change of mitochondrial membrane potential. The expression of caspase-3, 8, 9, Bax and Bcl-2 were examined by RT-PCR and Western blotting. Results The efficiency of gene silencing of lentivirus-mediated shRNA4 was up to 60%, compared with the parental and control groups. The viability of C neurons, together with mitochondrial membrane potential and expression of caspase-3, 8, 9, Bax/Bcl-2 was not influenced by lysine, even when the concentration was 10mmol/L. The viability of NC neurons was significantly higher than GCDH neurons, when with 5mmol/L lysine interference. When without lysine, there is no difference between the two. Moreover, 5mmol/L lysine could induce GCDH neurons apoptosis, and 10mmmol/L lysine could induce NC neurons apoptosis. In these apoptotic neurons, the mitochondrial membrane potential decrased, the expressions of caspase-3, 8, 9, Bax all increased significantly and Bcl-2 expression decreased, compared to normal cells.

β-Adrenergic-stimulated l-type channel Ca2 + entry mediates hypoxic Ca2 + overload in intact heart

Ca2+ mishandling plays a key role in ischemia- and hypoxia-related cardiac dysfunction and injury. However, the cellular and molecular mechanisms underlying hypoxic intracellular Ca2+ ([Ca2+]i) overload remain incompletely understood. This study aimed to investigate possible mechanisms of [Ca2+]i overload during hypoxia in the intact heart. In Langendorff-perfused heart expressing the Ca2+ indicator GCaMP2, confocal microscopy was used to simultaneously visualize [Ca2+]i, mitochondrial membrane potential (ΔΨm, by tetramethylrhodamine methyl ester) and sarcolemmal integrity (by Evans blue). Upon hypoxia (pO2 ~20mmHg in glucose-free perfusate), [Ca2+]i transients were initially enhanced and then became depressed, arrhythmic, and completely abolished within 12min. At ~20min, basal [Ca2+]i rose to its first peak at a supraphysiological level, coincident with loss of ΔΨm and onset of rigor. A greater [Ca2+]i rise occurred at ~2h and was linked to the loss of sarcolemmal integrity. Removal of extracellular Ca2+ or blockade of the l-type Ca2+ channel (LTCC) (10μM diltiazem or nifedipine) prevented [Ca2+]i overload and markedly delayed the loss of ΔΨm; by contrast, depletion of the sarcoplasmic reticulum Ca2+ store by thapsigargin did not have any significant effect. Importantly, β-adrenergic blockade or depletion of the sympathetic catecholamine store by reserpine slowed the Ca2+ and mitochondrial responses to hypoxia in intact heart. This LTCC-mediated hypoxic [Ca2+]i overload was reproduced in isolated cardiomyocytes when β-adrenergic agonist was present. Taken together, we conclude that Ca2+ entry through β-adrenergic-stimulated LTCC underlies hypoxia-induced [Ca2+]i overload and the ensuing loss of mitochondrial function in intact heart.