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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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This figure shows several parameters of intracellular free calcium transient such as (a) amplitude of calcium transients, (b) diastolic calcium levels, (c) changes of calcium transient decay constant, and (d) cell-shortening measurements from recordings from control and from virus infected myocytes after 1 to 3 days long culture. Bars represent means  ± SEM, and n represents the number of experiments.
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fig6: This figure shows several parameters of intracellular free calcium transient such as (a) amplitude of calcium transients, (b) diastolic calcium levels, (c) changes of calcium transient decay constant, and (d) cell-shortening measurements from recordings from control and from virus infected myocytes after 1 to 3 days long culture. Bars represent means ± SEM, and n represents the number of experiments.

Mentions: Cultured myocytes were stimulated at a constant frequency of 1 Hz through a pair of platinum electrodes. Similarly to Ito measurements, the steady-state [Ca2+]i transient and contractile function were measured and compared on a daily basis both in the infected and control cell populations. Original recordings of [Ca2+]i transients and cell shortening before and after virus infection are presented in Figure 5. The calcium transient kinetics were significantly distorted in culture, but were also not significantly altered by the virus-infection. As Figure 6 summarizes, we found no statistically significant differences in either the amplitude of calcium transient (A) or diastolic calcium level (B) between the studied groups. Moreover, transient decay time (C) in noninfected control myocytes was significantly larger than in the virus infected group after two and three days in culture. Conversely in virus-infected group's decay time was similar to freshly isolated cells. The cell-shortening measurements (D) represent a significant decline in noninfected cell culture after 1 and 3 days. Data indicate no significant changes in these parameters.


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)

This figure shows several parameters of intracellular free calcium transient such as (a) amplitude of calcium transients, (b) diastolic calcium levels, (c) changes of calcium transient decay constant, and (d) cell-shortening measurements from recordings from control and from virus infected myocytes after 1 to 3 days long culture. Bars represent means  ± SEM, and n represents the number of experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: This figure shows several parameters of intracellular free calcium transient such as (a) amplitude of calcium transients, (b) diastolic calcium levels, (c) changes of calcium transient decay constant, and (d) cell-shortening measurements from recordings from control and from virus infected myocytes after 1 to 3 days long culture. Bars represent means ± SEM, and n represents the number of experiments.
Mentions: Cultured myocytes were stimulated at a constant frequency of 1 Hz through a pair of platinum electrodes. Similarly to Ito measurements, the steady-state [Ca2+]i transient and contractile function were measured and compared on a daily basis both in the infected and control cell populations. Original recordings of [Ca2+]i transients and cell shortening before and after virus infection are presented in Figure 5. The calcium transient kinetics were significantly distorted in culture, but were also not significantly altered by the virus-infection. As Figure 6 summarizes, we found no statistically significant differences in either the amplitude of calcium transient (A) or diastolic calcium level (B) between the studied groups. Moreover, transient decay time (C) in noninfected control myocytes was significantly larger than in the virus infected group after two and three days in culture. Conversely in virus-infected group's decay time was similar to freshly isolated cells. The cell-shortening measurements (D) represent a significant decline in noninfected cell culture after 1 and 3 days. Data indicate no significant changes in these parameters.

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