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Herpesvirus-mediated delivery of a genetically encoded fluorescent Ca(2+) sensor to canine cardiomyocytes.

Prorok J, Kovács PP, Kristóf AA, Nagy N, Tombácz D, Tóth JS, Ordög B, Jost N, Virág L, Papp JG, Varró A, Tóth A, Boldogkoi Z - J. Biomed. Biotechnol. (2009)

Bottom Line: We demonstrated that the transfer efficiency of troponeon to cultured adult cardiac myocytes was virtually 100%.We showed that even after four days neither the amplitude nor the kinetics of the I(to) current was significantly changed and no major shifts occurred in parameters of [Ca(2+)](i) transients.Furthermore, we demonstrated that infection of cardiomyocytes with the virus did not affect the morphology, viability, and physiological attributes of cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Pharmacotherapy, University of Szeged, Hungary.

ABSTRACT
We report the development and application of a pseudorabies virus-based system for delivery of troponeon, a fluorescent Ca(2+) sensor to adult canine cardiomyocytes. The efficacy of transduction was assessed by calculating the ratio of fluorescently labelled and nonlabelled cells in cell culture. Interaction of the virus vector with electrophysiological properties of cardiomyocytes was evaluated by the analysis of transient outward current (I(to)), kinetics of the intracellular Ca(2+) transients, and cell shortening. Functionality of transferred troponeon was verified by FRET analysis. We demonstrated that the transfer efficiency of troponeon to cultured adult cardiac myocytes was virtually 100%. We showed that even after four days neither the amplitude nor the kinetics of the I(to) current was significantly changed and no major shifts occurred in parameters of [Ca(2+)](i) transients. Furthermore, we demonstrated that infection of cardiomyocytes with the virus did not affect the morphology, viability, and physiological attributes of cells.

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(a) Original recordings from cells expressed troponeon at the excitation wavelength of (a) 485 and 535 nm and (b) the characterized calcium transient by ratio of the fluorescence intensity (FCFP 485 / FCITRINE 535) from one day after infection.
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fig7: (a) Original recordings from cells expressed troponeon at the excitation wavelength of (a) 485 and 535 nm and (b) the characterized calcium transient by ratio of the fluorescence intensity (FCFP 485 / FCITRINE 535) from one day after infection.

Mentions: Changes in [Ca2+]i levels were characterized by the ratio of the emitted fluorescence intensities obtained at 480 and 535 nm wavelengths, (FFRET_CITRINE 535/ FCFP 480) following optical signal correction steps for photobleaching and nonspecific background fluorescence. Photobleaching leads to a steady decrease in the FRET ratio over time, because citrine is less photostable than CFP. To correct for bleaching, we multiplied the intensity of the CITRINE channel by a correction factor calculated from the intensity shift between two time points. For background correction we subtracted from the optical signals the nonspecific background fluorescence determined at both wavelengths by moving off the cell from the light path: FRATIO_corr = (F − F0)CITRINE / (F − F0)CFP. The background-corrected fluorescence ratio versus time curve can be considered as a representation of the intracellular Ca2+ transients Figure 7.


Herpesvirus-mediated delivery of a genetically encoded fluorescent Ca(2+) sensor to canine cardiomyocytes.

Prorok J, Kovács PP, Kristóf AA, Nagy N, Tombácz D, Tóth JS, Ordög B, Jost N, Virág L, Papp JG, Varró A, Tóth A, Boldogkoi Z - J. Biomed. Biotechnol. (2009)

(a) Original recordings from cells expressed troponeon at the excitation wavelength of (a) 485 and 535 nm and (b) the characterized calcium transient by ratio of the fluorescence intensity (FCFP 485 / FCITRINE 535) from one day after infection.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2712641&req=5

fig7: (a) Original recordings from cells expressed troponeon at the excitation wavelength of (a) 485 and 535 nm and (b) the characterized calcium transient by ratio of the fluorescence intensity (FCFP 485 / FCITRINE 535) from one day after infection.
Mentions: Changes in [Ca2+]i levels were characterized by the ratio of the emitted fluorescence intensities obtained at 480 and 535 nm wavelengths, (FFRET_CITRINE 535/ FCFP 480) following optical signal correction steps for photobleaching and nonspecific background fluorescence. Photobleaching leads to a steady decrease in the FRET ratio over time, because citrine is less photostable than CFP. To correct for bleaching, we multiplied the intensity of the CITRINE channel by a correction factor calculated from the intensity shift between two time points. For background correction we subtracted from the optical signals the nonspecific background fluorescence determined at both wavelengths by moving off the cell from the light path: FRATIO_corr = (F − F0)CITRINE / (F − F0)CFP. The background-corrected fluorescence ratio versus time curve can be considered as a representation of the intracellular Ca2+ transients Figure 7.

Bottom Line: We demonstrated that the transfer efficiency of troponeon to cultured adult cardiac myocytes was virtually 100%.We showed that even after four days neither the amplitude nor the kinetics of the I(to) current was significantly changed and no major shifts occurred in parameters of [Ca(2+)](i) transients.Furthermore, we demonstrated that infection of cardiomyocytes with the virus did not affect the morphology, viability, and physiological attributes of cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Pharmacotherapy, University of Szeged, Hungary.

ABSTRACT
We report the development and application of a pseudorabies virus-based system for delivery of troponeon, a fluorescent Ca(2+) sensor to adult canine cardiomyocytes. The efficacy of transduction was assessed by calculating the ratio of fluorescently labelled and nonlabelled cells in cell culture. Interaction of the virus vector with electrophysiological properties of cardiomyocytes was evaluated by the analysis of transient outward current (I(to)), kinetics of the intracellular Ca(2+) transients, and cell shortening. Functionality of transferred troponeon was verified by FRET analysis. We demonstrated that the transfer efficiency of troponeon to cultured adult cardiac myocytes was virtually 100%. We showed that even after four days neither the amplitude nor the kinetics of the I(to) current was significantly changed and no major shifts occurred in parameters of [Ca(2+)](i) transients. Furthermore, we demonstrated that infection of cardiomyocytes with the virus did not affect the morphology, viability, and physiological attributes of cells.

Show MeSH
Related in: MedlinePlus