A system for optical high resolution screening of electrical excitable cells

Müller, Oliver; Tian, Qi; Zantl, Roman; Kahl, Valentin; Lipp, Peter; Kaestner, Lars

doi:10.1016/j.ceca.2009.11.012

Cited by 15 publications

(9 citation statements)

References 23 publications

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“…This will allow transfer of our approach to precise cardiac safety screens according to FDA and EMA requirements [1,2]. We have recently demonstrated the possibility of an integration of adult cardiomyocytes into a high-content screening compatible workflow [27]. However, neonatal cardiomyocytes are a popular cell model, especially because cell isolation and handling is much easier compared to the adult situation.…”

Section: Discussionmentioning

confidence: 99%

Optical Action Potential Screening on Adult Ventricular Myocytes as an Alternative QT-screen

Tian

Oberhofer

Ruppenthal

et al. 2011

Cell Physiol Biochem

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Background/Aims: QT-interval screens are increasingly important for cardiac safety on all new medications. So far, investigations rely on animal experiments or cell-based screens solely probing for conductance alterations in heterologously expressed hERG-channels in cell lines allowing for a high degree of automation. Adult cardiomyocytes can not be handled by automated patch-clamp setups. Therefore optical screening of primary isolated ventricular myocytes is regarded as an alternative. Several optical voltage sensors have been reported for ratiometric measurements, but they all influenced the naïve action potential. The aim of the present study was to explore the recording conditions and define settings that allow optical QT-interval screens. Methods: Based on an improved optical design, individual action potentials could be recorded with an exceptional signal-to-noise-ratio. The sensors were validated using the patch-clamp technique, confocal microscopy and fluorescence lifetime imaging in combination with global unmixing procedures. Results: We show that the small molecule dye di-8-ANEPPS and the novel genetically encoded sensor Mermaid provide quantitative action potential information. When applying such sensors we identified distinctly different pharmacological profiles of action potentials for adult and neonatal rat cardiomyocytes. Conclusion: Optical methods can be used for QT-interval investigations based on cellular action potentials using either the small molecule dye di-8-ANEPPS or the genetically encoded sensor Mermaid. Adult cardiomyocytes are superior to neonatal cardiomyocytes for such pharmacological investigations. Optical QT-screens may replace intricate animal experiments.

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Section: Discussionmentioning

confidence: 99%

Optical Action Potential Screening on Adult Ventricular Myocytes as an Alternative QT-screen

Tian

Oberhofer

Ruppenthal

et al. 2011

Cell Physiol Biochem

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“…Although pharmaceutical by guest on May 11, 2018 http://circres.ahajournals.org/ Downloaded from or cardiovascular safety screens based on genetically encoded Ca 2+ sensors expressed in cardiomyocytes have never been published, the enabling technology is well described. 120,121 A robust isolation procedure 122 and greatly improved cell culture 23 enable the use of adult cardiomyocytes, which is a prerequisite that seems to be compulsory, particularly in consideration of the different gene expression patterns of embryonic, neonatal, and adult cardiac myocytes. 31,123 The major limitation is the requirement for electrodes in multiwell plates, which can be expensive; current designs are limited to 24-well plates.…”

Section: Pharmaceutical Screening Based On Cardiac Myocytesmentioning

confidence: 99%

“…31,123 The major limitation is the requirement for electrodes in multiwell plates, which can be expensive; current designs are limited to 24-well plates. 84,121 However, the counterparts of GECI and genetically encoded optical manipulators, such as channel rhodopsins, have been successfully used in the heart. 124 These molecules could provide an alternative means for the electric stimulation of isolated adult cardiac myocytes without electrodes and could thus enable the use of 384-well plates.…”

Section: Pharmaceutical Screening Based On Cardiac Myocytesmentioning

confidence: 99%

Genetically Encoded Ca ²⁺ Indicators in Cardiac Myocytes

Kaestner

Scholz

Tian

et al. 2014

Circulation Research

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“…Isolated myocytes were loaded with fluo-4 AM as previously described 13 (1.0 mol/L for 30 minutes with 15 minutes for deesterification) and imaged with a resonant confocal scanner (Leica TCS SP5, Leica Microsystems GmbH, Wetzlar, Germany) at a frame rate of approximately 150 Hz. For details of solution composition and optical setup as well as for data preparation and calculation of Ca 2ϩ concentrations, please refer to the Online Supplement.…”

Section: High-resolution High-speed Confocal Imagingmentioning

confidence: 99%

Noise-Free Visualization of Microscopic Calcium Signaling by Pixel-Wise Fitting

Tian

Kaestner

Lipp

2012

Circulation Research

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Rationale: Our insights into physiological and pathophysiological cardiac excitation-contraction coupling has greatly benefited from significant advancement in optical technologies such as high-speed confocal microscopy. This has pushed pixel dwell times into the time domain of nanoseconds, resulting in low signal-to-noise ratios, which have limited data analysis and interpretation.Objective: Line scan imaging has been and still is dominant in high speed confocal recording. It allows analysis only of a small fraction of a cell's cross section (1.5%), but the appreciation of spatiotemporal fine details of excitation-contraction coupling is instrumental for the further understanding of pathological mechanisms. We aim to provide a novel analysis tool to extract otherwise hidden fine details in cardiac excitation-contraction coupling from high-speed 2-dimensional confocal image series. Methods and Results:We demonstrate that high-speed 2-dimensional confocal data (150 frames/s) can be analyzed quantitatively by a pixel-wise fitting approach, using a mathematical formalism to phenomenologically describe local calcium transients. Such an approach produces virtually noise-free fluorescence data originating from minute volumes (0.025 femtoliter) and allows extraction of detailed and most importantly quantitative and mechanistically novel information on microscopic calcium signaling and excitation-contraction coupling in a robust manner. Conclusions:Pixel-wise fitting provides novel insights into cardiac excitation-contraction coupling. Specifically, it revealed microscopic calcium alternans on the level of individual coupling sites. Microscopic calcium alternans is an early precursor of cellular alternans and as such will shed more light onto this mechanism leading to cardiac arrhythmia. (Circ Res. 2012;111:17-27.) Key Words: excitation-contraction coupling Ⅲ calcium-induced calcium release Ⅲ noise-free image sequences Ⅲ alternans Ⅲ arrhythmogenic precursor Ⅲ Ca 2ϩ transients H igh-resolution live-cell imaging of subcellular signaling events has greatly fostered our understanding of cardiac physiology and pathophysiology. 1 In this, a major driving force was the advancement in laser-scanning technology that enabled ultrafast confocal imaging (Ͼ100 frames/s) without compromising the optical resolution. 2 Although researchers are technically able to follow fast subcellular signaling, such as excitation-contraction coupling (ECC) in cardiac myocytes, 3 decreasing the single pixel dwell times and concomitant decreases in the signal-to-noise ratio (SNR) have limited the progress. However, scientific interest is not limited to the occurrence of Ca 2ϩ induced Ca 2ϩ release (CICR) per se but also focuses on where, how much, and how fast the Ca 2ϩ increases occur because the signaling information is encoded in all of these properties. 4 Although cardiac Ca 2ϩ transients appear homogeneous under physiological conditions, they arise from the coordinated activity of many microscopic ECC units, 3 comprising voltage operated Ca 2ϩ ch...

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A system for optical high resolution screening of electrical excitable cells

Cited by 15 publications

References 23 publications

Optical Action Potential Screening on Adult Ventricular Myocytes as an Alternative QT-screen

Optical Action Potential Screening on Adult Ventricular Myocytes as an Alternative QT-screen

Genetically Encoded Ca ²⁺ Indicators in Cardiac Myocytes

Noise-Free Visualization of Microscopic Calcium Signaling by Pixel-Wise Fitting

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A system for optical high resolution screening of electrical excitable cells

Cited by 15 publications

References 23 publications

Optical Action Potential Screening on Adult Ventricular Myocytes as an Alternative QT-screen

Optical Action Potential Screening on Adult Ventricular Myocytes as an Alternative QT-screen

Genetically Encoded Ca 2+ Indicators in Cardiac Myocytes

Noise-Free Visualization of Microscopic Calcium Signaling by Pixel-Wise Fitting

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Genetically Encoded Ca ²⁺ Indicators in Cardiac Myocytes