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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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Representative low- and high-magnification transillumination of adult dog myocytes after isolation (day 0) and 1–3 days of culture (day 1, day 3). (a) shows the yield of the myocytes before and one day after culturing and infection. The high-magnification transillumination (b) shows the morphology of adult dog myocytes after isolation (day 0) and 1–3 day of culture (day 1, day 3) and the morphological changes of living cells, versus the culturing time from noninfected (right) and the virus-infected groups (left). After one day plated myocytes more than 80% displayed a rod-shaped morphology and healthy cross-striation. After 3 days (day 3) in culture, cells remained rod-shaped and partially cross-striated, and the main change was that cell ends became progressively more rounded.
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fig2: Representative low- and high-magnification transillumination of adult dog myocytes after isolation (day 0) and 1–3 days of culture (day 1, day 3). (a) shows the yield of the myocytes before and one day after culturing and infection. The high-magnification transillumination (b) shows the morphology of adult dog myocytes after isolation (day 0) and 1–3 day of culture (day 1, day 3) and the morphological changes of living cells, versus the culturing time from noninfected (right) and the virus-infected groups (left). After one day plated myocytes more than 80% displayed a rod-shaped morphology and healthy cross-striation. After 3 days (day 3) in culture, cells remained rod-shaped and partially cross-striated, and the main change was that cell ends became progressively more rounded.

Mentions: Using the described method for isolation of adult dog left ventricular myocytes, we routinely obtained a high yield (more than 80%) and high (more than 80%) percentage of rod-shaped myocytes that were suitable not only for acute functional studies but, more importantly, for short-term culture and gene transfer. Figure 2(a) shows a transillumination image of freshly isolated and 1-day-old cultured cardiomyocytes from the left ventricle. To establish optimal surviving conditions several culture conditions were tested based on microscopic evaluation of changes in cellular morphology during the four days of culture. Cultured cells were used 1–3 days after isolation. During this period, visible small-scale changes in cell shape and cross-striation could be observed. Figure 2(b) shows representative photographs of canine myocytes over time in culture. Features typical of acutely isolated (Day 0) cells were the rod shape with rectangular stepped ends and clear cross-striations. After 1 day (Day 1) in culture, the cells were still rod-shaped with clear cross-striations; however, the ends of the cells started to become slightly rounded in appearance. After 3 days (Day 3) in culture, cells remained rod-shaped and cross-striated, and the main change was that cell ends became progressively more rounded (see Table 1 for cell survivals).


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)

Representative low- and high-magnification transillumination of adult dog myocytes after isolation (day 0) and 1–3 days of culture (day 1, day 3). (a) shows the yield of the myocytes before and one day after culturing and infection. The high-magnification transillumination (b) shows the morphology of adult dog myocytes after isolation (day 0) and 1–3 day of culture (day 1, day 3) and the morphological changes of living cells, versus the culturing time from noninfected (right) and the virus-infected groups (left). After one day plated myocytes more than 80% displayed a rod-shaped morphology and healthy cross-striation. After 3 days (day 3) in culture, cells remained rod-shaped and partially cross-striated, and the main change was that cell ends became progressively more rounded.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Representative low- and high-magnification transillumination of adult dog myocytes after isolation (day 0) and 1–3 days of culture (day 1, day 3). (a) shows the yield of the myocytes before and one day after culturing and infection. The high-magnification transillumination (b) shows the morphology of adult dog myocytes after isolation (day 0) and 1–3 day of culture (day 1, day 3) and the morphological changes of living cells, versus the culturing time from noninfected (right) and the virus-infected groups (left). After one day plated myocytes more than 80% displayed a rod-shaped morphology and healthy cross-striation. After 3 days (day 3) in culture, cells remained rod-shaped and partially cross-striated, and the main change was that cell ends became progressively more rounded.
Mentions: Using the described method for isolation of adult dog left ventricular myocytes, we routinely obtained a high yield (more than 80%) and high (more than 80%) percentage of rod-shaped myocytes that were suitable not only for acute functional studies but, more importantly, for short-term culture and gene transfer. Figure 2(a) shows a transillumination image of freshly isolated and 1-day-old cultured cardiomyocytes from the left ventricle. To establish optimal surviving conditions several culture conditions were tested based on microscopic evaluation of changes in cellular morphology during the four days of culture. Cultured cells were used 1–3 days after isolation. During this period, visible small-scale changes in cell shape and cross-striation could be observed. Figure 2(b) shows representative photographs of canine myocytes over time in culture. Features typical of acutely isolated (Day 0) cells were the rod shape with rectangular stepped ends and clear cross-striations. After 1 day (Day 1) in culture, the cells were still rod-shaped with clear cross-striations; however, the ends of the cells started to become slightly rounded in appearance. After 3 days (Day 3) in culture, cells remained rod-shaped and cross-striated, and the main change was that cell ends became progressively more rounded (see Table 1 for cell survivals).

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