Tetramethylrhodamine Methylester Fluorescence Research Articles

Abstract 10172: Mitochondrial Connexin43 Can be Involved in the Occurrence of Arrhythmias

Background: Connexin43 (Cx43) forms gap junctions in cardiac ventricles and exits as hemichannels in the inner mitochondrial membrane (mCx43). Objective: To examine whether mCx43 is involved in the occurrence of arrhythmias with modulation of mitochondrial K ATP channels (mK ATP ). Methods: To generate cardiac-specific Cx43-deficient ( Cx43 -/- ) mice, Cx43 flox/flox mice were crossed with α-myosin heavy chain -cre +/- mice. The resulting offspring, Cx43 -/- -mice and their littermates ( Cx43 +/+ -mice) were used. Trabeculae were obtained from right ventricles. Force was measured with a strain gauge, Ca 2+ within the mitochondria (Ca m ) with rhod-2, intracellular Ca 2+ (Ca i ) with microinjected fura-2 (22°C). The minimal extracellular Ca 2+ concentration (Cao min ), at which arrhythmias were induced by electrical stimulation, was determined. Using isolated single ventricular myocytes, mitochondrial membrane potential (ΔΨ m ) was estimated with tetramethylrhodamine methylester (TMRM), ROS production with 2’,7’-dichlorofluorescein (DCF), and Ca 2+ spark frequency with fluo-4 and confocal microscopy. Results: Cx43 -/- -mice showed sudden death within 8 weeks (p<0.01). Cx43 was present in the inner mitochondrial membrane in Cx43 +/+ -mice but not in Cx43 -/- -mice. Developed force and Ca i transients by 0.5 Hz electrical stimulation showed no difference between Cx43 -/- - and Cx43 +/+ -mice (n=7). The Cao min in Cx43 -/- -mice was lower than that in Cx43 +/+ -mice (n=5, p<0.01) and was increased by diazoxide (DZX, n=4, p<0.05), suggesting that arrhythmia susceptibility is increased in Cx43 -/- -mice and is suppressed by DZX. Ca 2+ spark frequency and DCF oxidation rate in Cx43 -/- -mice were higher (p<0.01) and were decreased by DZX (p<0.01). TMRM fluorescence was decreased in Cx43 -/- -mice, and it was increased by DZX (p<0.01) and was decreased by 5-hydroxydecanoic acid (p<0.01). Rhod-2 signal was increased at 4 - 6 mM extracellular Ca 2+ in Cx43 -/- -mice but not in Cx43 +/+ -mice. Conclusions: Cx43 deficiency depolarizes ΔΨ m and increases Ca m at higher Ca o , ROS production, Ca 2+ spark frequency, and arrhythmia susceptibility. These changes in Cx43 -/- -mice are suppressed by DZX. Thus, mCx43 can be involved in the occurrence of arrhythmias with modulation of mK ATP .

B-PO02-019 MITOCHONDRIAL CONNEXIN43 IS INVOLVED IN THE OCCURRENCE OF ARRHYTHMIAS WITH MODULATION OF MITOCHONDRIAL KATP CHANNELS

Connexin43 (Cx43) forms gap junctional channels in cardiac ventricles and exits as hemichannels in the inner mitochondrial membrane (mCx43). To examine whether mCx43 and mitochondrial KATP channels (mKATP) affect the occurrence of arrhythmias. To generate cardiac-specific Cx43-deficient (Cx43-/-) mice, Cx43flox/flox mice were crossed with α-myosin heavy chain (Myh6)-cre+/- mice. The resulting offspring, Cx43-/--mice and their littermates (Cx43+/+-mice) were used. Trabeculae were dissected from ventricles. Force was measured with a silicon strain gauge, Ca2+ within the mitochondria (Cam) with rhod-2, intracellular Ca2+ (Cai) with microinjected fura-2 (22°C). The minimal extracellular Ca2+ concentration (Caomin), at which arrhythmias were induced by electrical stimulation, was determined. Using isolated single ventricular myocytes, mitochondrial membrane potential (ΔΨm) was estimated with tetramethylrhodamine methylester (TMRM), ROS production with 2’,7’-dichlorofluorescein (DCF), and Ca2+ spark frequency with fluo-4 and confocal microscopy. Most of Cx43-/--mice died within 8 weeks (p<0.01). Cx43 was present in the inner mitochondrial membrane in Cx43+/+-mice but not in Cx43-/--mice. Developed force and Cai transients by 0.5 Hz electrical stimulation showed no difference between Cx43-/-- and Cx43+/+-mice (n=7). The Caomin in Cx43-/--mice was lower than that in Cx43+/+-mice (n=5, p<0.01) and was increased by diazoxide (DZX, n=4, p<0.05), suggesting that arrhythmogenesis is increased in Cx43-/--mice and is suppressed by DZX. Ca2+ spark frequency and DCF oxidation rate in Cx43-/--mice were higher (p<0.01) and were decreased by DZX (p<0.01). TMRM fluorescence was decreased after 1 Hz stimulation in Cx43-/--mice, and it was increased by DZX (p<0.01) and was decreased by 5-hydroxydecanoic acid (p<0.01). Rhod-2 signal was increased in Cx43-/--mice but not in Cx43+/+-mice at 4 - 6 mM extracellular Ca2+ (Cao). Cx43 deficiency depolarizes ΔΨm and increases Cam at higher Cao, ROS production, Ca2+ spark frequency, and arrhythmogenesis. All these changes in Cx43-/--mice are suppressed by DZX. Thus, with modulation of mKATP, mCx43 can be involved in the occurrence of arrhythmias.

Mitochondrial connexin43 affects arrhythmogenesis with modulation of mitochondrial KATP channels

Abstract Introduction Connexin43 (Cx43) forms gap junction channels in ventricular muscle and also exits in the inner mitochondrial membrane (mCx43). We have previously reported that carbenoxolone, a blocker of Cx43, increased the occurrence of arrhythmias with modulation of mitochondrial KATP channels (mKATP). Purpose To examine how mCx43 and mKATP affect arrhythmogenesis, using cardiac-specific Cx43-deficient (Cx43−/−) mice. Methods To generate cardiac-specific Cx43−/− mice, Cx43flox/flox mice were crossed with α-myosin heavy chain (Myh6)-cre+/− mice. The resulting offspring, Cx43flox/flox/ Myh6-cre+/− mice (Cx43−/− mice) and their littermates (Cx43+/+ mice), were used. Trabeculae were dissected from right ventricles of mouse hearts (length ∼0.9 mm, width ∼200 μm, thickness ∼80 μm). Force was measured with a strain gauge, and intracellular Ca2+ (Cai) with fura-2 and a CCD camera (22°C). To assess arrhythmogenesis, the minimal extracellular Ca2+ concentration (Caomin), at which arrhythmias were induced by electrical stimulation (0.3-s stimulus intervals, 100 nM isoproterenol), was determined in mouse trabeculae. Using isolated single ventricular myocytes, mitochondrial membrane potential (ΔΨm) was estimated with tetramethylrhodamine methylester (TMRM) fluorescence, ROS production was estimated with 2',7'-dichlorofluorescein (DCF) fluorescence, and Ca2+ spark frequency was measured with fluo-4 and confocal microscopy. To investigate the roles of mKATP, 0.1 mM diazoxide, a mKATP channel opener, and 0.2 mM 5-hydroxydecanoic acid (5-HD), a mKATP channel inhibitor, were used. Results Most of Cx43−/− mice suddenly died within 8 weeks (p&amp;lt;0.01). Cx43 was present in the inner mitochondrial membrane in Cx43+/+ mice, while it was not present in Cx43−/− mice. The resting and developed force and Cai transients by electrical stimulation (2-s stimulus intervals) showed no difference between Cx43−/− and Cx43+/+ mouse trabeculae (n=7). The Caomin in Cx43−/− mice was lower than that in Cx43+/+ mice (p&amp;lt;0.01), and diazoxide increased the Caomin in Cx43−/− mice (p&amp;lt;0.01), suggesting that arrhythmogenesis is increased in Cx43−/− mice and is decreased by diazoxide. Ca2+ spark frequency and DCF oxidation rate in Cx43−/− mice were higher than those in Cx43+/+ mice (p&amp;lt;0.01). They were decreased by diazoxide and were increased by 5-HD (p&amp;lt;0.01). TMRM fluorescence was decreased after electrical stimulation (1-s stimulus intervals) in Cx43−/− mice and was further decreased by 5-HD (p&amp;lt;0.01), suggesting depolarization of ΔΨm in Cx43−/− mice. Such decrease in TMRM fluorescence in Cx43−/− mice was suppressed by diazoxide and did not occur in Cx43+/+ mice. Conclusions These results suggest that with modulation of mKATP, mCx43 plays important roles in ΔΨm determination, ROS production, and Ca2+ leak from the sarcoplasmic reticulum, thereby affecting arrhythmogenesis. Therefore, it is likely that mCx43 and mKATP could become a therapeutic target for controlling arrythmias. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science

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.

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

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.

Voltage Dependent Anion Channel-3 (VDAC3) is the Major Isoform Contributing to Mitochondrial Metabolism in HepG2 Cells and is Regulated by Free Tubulin and Erastin

BACKGROUND: VDAC controls the flux of hydrophilic metabolites into mitochondria. In vitro, tubulin closes VDAC and in situ free tubulin dynamically modulates ΔΨ. Erastin, a ligand for VDAC, increases ΔΨ in HepG2 cells. Here, we hypothesize that VDAC regulates mitochondrial metabolism and that free tubulin decreases and erastin increases VDAC conductance. Our AIM was to determine ΔΨ, ATP, NADH, and response to free tubulin and erastin after VDAC knockdown (KD). METHODS: HepG2 cells were transfected with siRNAs (5 nM, Ambion) against VDAC1/2/3. At 48 h after transfection, ΔΨ was assessed by fluorescence of tetramethylrhodamine methylester (TMRM) and NADH by autofluorescence using confocal/multiphoton microscopy. Fluorescent beads were fiduciary markers. Adenine nucleotides were determined by HPLC. RESULTS: siRNA decreased mRNA and protein for each VDAC isoform by ∼90%. Double KD of VDAC1/2, VDAC1/3 and VDAC2/3 decreased TMRM fluorescence by ∼20, 55 and 73%, respectively, compared to 100% for non-target siRNA. In reconstituted bilayers, VDAC from HepG2 formed typical anion selective and voltage-gated channels reversibly blocked by dimeric tubulin. Nocodazole decreased ΔΨ by ∼61% in non-target cells and 43, 14 and 17% after KD of VDAC1/2, VDAC1/3 and VDAC2/3. VDAC3 KD decreased ATP by ∼48%, total adenine nucleotides by ∼45%, NADH by ∼33% and the NADH/NAD ratio by ∼60%. Erastin increased ΔΨ by ∼46-48% in non-target and VDAC1/2 KD cells but failed to return ΔΨ to baseline levels after VDAC1/3 and VDAC2/3 KD. Erastin also blocked and reversed depolarization induced by nocodazole. CONCLUSION: VDAC3, the least abundant isoform, contributes most to maintenance of mitochondrial metabolism in HepG2 cells. Erastin antagonizes the inhibitory effect of free tubulin. These results indicate that VDAC, especially VDAC3, regulates mitochondrial metabolism in hepatoma cells.

Knockdown of VDAC Isoforms Decreases Mitochondrial Membrane Potential and Modulates the Effects of Free Tubulin in HepG2 Hepatoma Cells

BACKGROUND: Mitochondrial membrane potential (ΔΨ) depends on flux of metabolites through voltage dependent anion channels (VDAC) in the mitochondrial outer membrane. In vitro, tubulin binding induces VDAC closure. In intact cells, nocodazole (Ncz) increases free tubulin and decreases ΔΨ, whereas paclitaxel (Ptx) promotes tubulin polymerization and increases ΔΨ. These effects are attributed to closing and opening of VDAC. Here, we hypothesize that VDAC knockdown will decrease ΔΨ and block the inhibitory effect of free tubulin on ΔΨ. METHODS: HepG2 cells were transfected with individual siRNAs for VDAC1/2/3 (5 nM, Ambion) for 48 h. To assess ΔΨ, fluorescence of tetramethylrhodamine methylester (TMRM) was measured by confocal microscopy. RESULTS: VDAC1/2/3 relative mRNA abundance by qPCR was ∼39, 50 and 10%, respectively. Each siRNA decreased its corresponding VDAC mRNA by ∼90% without affecting other isoforms. After VDAC1/2/3 knockdown, TMRM fluorescence was 58, 41 and 21%, respectively, compared to 100% after non-target siRNA. In control cells, Ncz decreased TMRM fluorescence by 70%, and Ptx increased fluorescence by 51%. After knockdown of VDAC1/2/3, Ncz decreased TMRM fluorescence by 25, 22 and 9%, respectively, whereas Ptx increased fluorescence by 26, 28, and 27% . CONCLUSION: Each of the three VDAC isoforms contributes to maintenance of ΔΨ in HepG2 cells. After knockdown, the relative response to changes of free tubulin was attenuated. These results are consistent with the conclusion that free tubulin dynamically regulates ΔΨ in HepG2 cells by interacting with VDAC.

Oxidant-induced inhibition of the plasma membrane Ca2+-ATPase in pancreatic acinar cells: role of the mitochondria

Impairment of the normal spatiotemporal pattern of intracellular Ca(2+) ([Ca(2+)](i)) signaling, and in particular, the transition to an irreversible "Ca(2+) overload" response, has been implicated in various pathophysiological states. In some diseases, including pancreatitis, oxidative stress has been suggested to mediate this Ca(2+) overload and the associated cell injury. We have previously demonstrated that oxidative stress with hydrogen peroxide (H(2)O(2)) evokes a Ca(2+) overload response and inhibition of plasma membrane Ca(2+)-ATPase (PMCA) in rat pancreatic acinar cells (Bruce JI and Elliott AC. Am J Physiol Cell Physiol 293: C938-C950, 2007). The aim of the present study was to further examine this oxidant-impaired inhibition of the PMCA, focusing on the role of the mitochondria. Using a [Ca(2+)](i) clearance assay in which mitochondrial Ca(2+) uptake was blocked with Ru-360, H(2)O(2) (50 microM-1 mM) markedly inhibited the PMCA activity. This H(2)O(2)-induced inhibition of the PMCA correlated with mitochondrial depolarization (assessed using tetramethylrhodamine methylester fluorescence) but could occur without significant ATP depletion (assessed using Magnesium Green fluorescence). The H(2)O(2)-induced PMCA inhibition was sensitive to the mitochondrial permeability transition pore (mPTP) inhibitors, cyclosporin-A and bongkrekic acid. These data suggest that oxidant-induced opening of the mPTP and mitochondrial depolarization may lead to an inhibition of the PMCA that is independent of mitochondrial Ca(2+) handling and ATP depletion, and we speculate that this may involve the release of a mitochondrial factor. Such a phenomenon may be responsible for the Ca(2+) overload response, and for the transition between apoptotic and necrotic cell death thought to be important in many disease states.

A novel butyrolactone derivative inhibited smooth muscle cell migration and proliferation and maintained endothelial cell functions through selectively affecting Na, K‐ATPase activity and mitochondria membrane potential during in vitro angiogenesis

We have found that 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran -2(3H)-one (3BDO), could effectively suppress human umbilical vascular endothelial cell (HUVEC) apoptosis induced by deprivation of fibroblast growth factor-2 and serum. Here, our purpose was to investigate whether 3BDO could modulate angiogenesis and its possible acting mechanism. The effect of 3BDO on angiogenesis was investigated by capillary-like tubule formation and rat aortic ring assay. Proliferation and migration of cells were detected by counting living cell number and scraping cell monolayer, respectively. Na, K-ATPase activity was measured spectrophotometrically. Mitochondrial membrane potential was analyzed using tetramethylrhodamine methylester fluorescence by confocal microscopy. Our results showed that 3BDO inhibited migration and proliferation of vascular smooth muscle cells (VSMCs), but maintained migration and tubule formation of HUVECs. In HUVECs, 3BDO inhibited Na, K-ATPase activity, but had no effect on mitochondria membrane potential. In VSMCs, it did not affect Na, K-ATPase activity, but depressed mitochondria membrane potential obviously. The data showed that 3BDO had selective effects on HUVECs and VSMCs, it might perform its role through the selective effects on the activity of Na, K-ATPase and the mitochondria membrane potential in HUVECs and VSMCs.

Role of oxidant stress in the permeability transition induced in rat hepatic mitochondria by hydrophobic bile acids.

Hydrophobic bile acids may cause hepatocellular necrosis and apoptosis during cholestatic liver diseases. The mechanism for this injury may involve mitochondrial dysfunction and the generation of oxidant stress. The purpose of this study was to determine the relationship of oxidant stress and the mitochondrial membrane permeability transition (MMPT) in hepatocyte necrosis induced by bile acids. The MMPT was measured spectrophotometrically and morphologically in rat liver mitochondria exposed to glycochenodeoxycholic acid (GCDC). Freshly isolated rat hepatocytes were exposed to GCDC and hepatocellular necrosis was assessed by lactate dehydrogenase release, hydroperoxide generation by dichlorofluorescein fluorescence, and the MMPT in cells by JC1 and tetramethylrhodamine methylester fluorescence on flow cytometry. GCDC induced the MMPT in a dose- and Ca(2+)-dependent manner. Antioxidants significantly inhibited the GCDC-induced MMPT and the generation of hydroperoxides in isolated mitochondria. Other detergents failed to induce the MMPT and a calpain-like protease inhibitor had no effect on the GCDC-induced MMPT. In isolated rat hepatocytes, GCDC induced the MMPT, which was inhibited by antioxidants. Blocking the MMPT in hepatocytes reduced hepatocyte necrosis and oxidant stress caused by GCDC. Oxidant stress, and not detergent effects or the stimulation of calpain-like proteases, mediates the GCDC-induced MMPT in hepatocytes. We propose that reducing mitochondrial generation of reactive oxygen species or preventing increases in mitochondrial Ca(2+) may protect the hepatocyte against bile acid-induced necrosis.

Effects of pacing and isoproterenol on mitochondrial Ca2+ transients in adult rabbit cardiac myocytes during the contractile cycle

Using laser scanning confocal microscopy, we have developed methods to measure mitochondrial free Ca2+ in cultured adult rabbit cardiac myocytes during excitation-contraction. Here, we examined the effect of isoproterenol and the rate of electrical pacing on the magnitude and duration of mitochondrial Ca transients. The mitochondria and cytosol of myocytes were loaded with the Ca2+-indicating fluorophore, Fluo-3-AM, at 4°C. Subsequently, the mitochondria were labeled with tetramethylrhodamine methylester (TMRM). In line scans, red TMRM fluorescence and green Fluo-3 fluorescence were simultaneously imaged by laser scanning confocal microscopy. Large transients of Fluo-3 fluorescence after electrical stimulation were observed both in cytosol and mitochondria (Figures 1). Isoproterenol (1 μM) increased the magnitude of Ca2+ transients caused by field stimulation and their subsequent rate of decay (Figure 2). This effect of isoproterenol was more marked in the cytosol than in mitochondria. In particular, the effect of isoproterenol on Ca2+ transients was least in perinuclear mitochondria.