Validation of a voltage-sensitive dye (di-4-ANEPPS)-based method for assessing drug-induced delayed repolarisation in Beagle dog left ventricular midmyocardial myocytes

Hardy, Matthew E.L.; Pollard, Chris; Small, Ben G; Bridgland-Taylor, Matthew; Woods, A.; Valentin, Jean‐Pierre; Abi‐Gerges, Najah

doi:10.1016/j.vascn.2009.03.005

Cited by 22 publications

(32 citation statements)

References 44 publications

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“…An alternative technique to study APs that is less invasive, requires less skill, and yields higher throughput than the traditional techniques is optical recording using a fast voltagesensitive dye (di-4-ANEPPS). 22 Analysis gives information on AP duration and changes therein, but the information is less detailed compared with the techniques just mentioned. Multi-electrode arrays are used to study the electrical activity of SC-CM clusters by recording a field potential.…”

Section: Current Modelsmentioning

confidence: 99%

Impedance-Based Detection of Beating Rhythm and Proarrhythmic Effects of Compounds on Stem Cell-Derived Cardiomyocytes

Jonsson

Wang

Becker

2011

ASSAY and Drug Development Technologies

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The xCELLigence real time cell analyzer Cardio system offers a new system for real-time cell analysis that measures impedance-based signals in a label-free noninvasive manner. The aim of this study was to test whether impedance readings are a useful tool to detect compound effects on beating frequency (beats per minute, bpm) and arrhythmias of human induced pluripotent stem cell-and a mouse embryonic stem cell-derived cardiomyocyte line (hiPSC-CM and mESC-CM, respectively). Baseline values for control wells were 45 -3 and 179 -6 bpm, respectively (n = 6). Correspondingly, isoproterenol increased beating frequency by 77% and 71%, whereas carbachol decreased frequency by 11% and 100% (stopped in 5/6 mESC-CM wells). E-4031 decreased beating rate and caused arrhythmias in both cell types, however, more pronounced in the human iPSC-CMs. Amlodipine inhibited contractions in both models, and T-type calcium channel block strongly reduced beating rate and eventually stopped beating in mESC-CM but caused a smaller effect in hiPSC-CM. The results of this initial study show that, under the right conditions, the beating frequency of a monolayer of cells can be stably recorded over several days. Additionally, the system detects changes in beating frequency and amplitude caused by added reference compounds. This assay system has the potential to enable medium-throughput screening, but for implementation into routine daily work, extended validation, testing of additional batches of cardiomyocytes, and further assay optimization (e.g., frequency of media exchange, growth matrix, seeding density, age of cells after plating, and temperature control) will be needed.

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Section: Current Modelsmentioning

confidence: 99%

Impedance-Based Detection of Beating Rhythm and Proarrhythmic Effects of Compounds on Stem Cell-Derived Cardiomyocytes

Jonsson

Wang

Becker

2011

ASSAY and Drug Development Technologies

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“…Prolongation of the action potential in isolated cardiomyocytes due to phototoxicity has been shown at concentrations as low as 0.3 μ M of di-4-ANEPPS. 2 Interestingly, this phenomenon has not been reported in intact cardiac tissue. However, in the isolated rat heart, Nygren et al 3 showed that di-4-ANEPPS prolongs the PQ interval and transiently blocks atrioventricular conduction during dye loading.…”

Section: Introductionmentioning

confidence: 96%

The voltage-sensitive dye di-4-ANEPPS slows conduction velocity in isolated guinea pig hearts

Larsen¹,

Sciuto²,

Moreno³

et al. 2012

Heart Rhythm

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BACKGROUND Voltage-sensitive dyes are important tools for mapping electrical activity in the heart. However, little is known about the effects of voltage-sensitive dyes on cardiac electrophysiology. OBJECTIVE To test the hypothesis that the voltage-sensitive dye di-4-ANEPPS modulates cardiac impulse propagation. METHODS Electrical and optical mapping experiments were performed in isolated Langendorff perfused guinea pig hearts. The effect of di-4-ANEPPS on conduction velocity and anisotropy of propagation was quantified. HeLa cells expressing connexin 43 were used to evaluate the effect of di-4-ANEPPS on gap junctional conductance. RESULTS In electrical mapping experiments, di-4-ANEPPS (7.5 μM) was found to decrease both longitudinal and transverse conduction velocities significantly compared with control. No change in the anisotropy of propagation was observed. Similar results were obtained in optical mapping experiments. In these experiments, the effect of di-4-ANEPPS was dose dependent. di-4-ANEPPS had no detectable effect on connexin 43–mediated gap junctional conductance in transfected HeLa cells. CONCLUSION Our results demonstrate that the voltage-sensitive dye di-4-ANEPPS directly and dose-dependently modulates cardiac impulse propagation. The effect is not likely mediated by connexin 43 inhibition. Our results highlight an important caveat that should be taken into account when interpreting data obtained using di-4-ANEPPS in cardiac preparations.

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“…11,13,18,19 However, it is usually less pronounced in multicellular aggregates 12,20 or tissue preparations.…”

Section: Discussionmentioning

confidence: 99%

“…11,19 Incubation of the cells with antioxidizing agents significantly reduces the manifestations of phototoxicity. 19 The differences in photo sensitivity between single cells and intact tissue can be explained by a lack in isolated cells of an efficient ROS scavenging system, which was damaged during the cell isolation.…”

Section: 821mentioning

confidence: 99%

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Optical mapping at increased illumination intensities

et al. 2012

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Abstract. Voltage-sensitive fluorescent dyes have become a major tool in cardiac and neuro-electrophysiology. Achieving high signal-to-noise ratios requires increased illumination intensities, which may cause photobleaching and phototoxicity. The optimal range of illumination intensities varies for different dyes and must be evaluated individually. We evaluate two dyes: di-4-ANBDQBS (excitation 660 nm) and di-4-ANEPPS (excitation 532 nm) in the guinea pig heart. The light intensity varies from 0.1 to 5 mW∕mm 2 , with the upper limit at 5 to 10 times above values reported in the literature. The duration of illumination was 60 s, which in guinea pigs corresponds to 300 beats at a normal heart rate. Within the identified duration and intensity range, neither dye shows significant photobleaching or detectable phototoxic effects. However, light absorption at higher intensities causes noticeable tissue heating, which affects the electrophysiological parameters. The most pronounced effect is a shortening of the action potential duration, which, in the case of 532-nm excitation, can reach ∼30%. At 660-nm excitation, the effect is ∼10%. These findings may have important implications for the design of optical mapping protocols in biomedical applications.

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Validation of a voltage-sensitive dye (di-4-ANEPPS)-based method for assessing drug-induced delayed repolarisation in Beagle dog left ventricular midmyocardial myocytes

Cited by 22 publications

References 44 publications

Impedance-Based Detection of Beating Rhythm and Proarrhythmic Effects of Compounds on Stem Cell-Derived Cardiomyocytes

Impedance-Based Detection of Beating Rhythm and Proarrhythmic Effects of Compounds on Stem Cell-Derived Cardiomyocytes

The voltage-sensitive dye di-4-ANEPPS slows conduction velocity in isolated guinea pig hearts

Optical mapping at increased illumination intensities

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