Herpesvirus-mediated delivery of a genetically encoded fluorescent Ca(2+) sensor to canine cardiomyocytes.
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.
Prorok
János
J
Department of Pharmacology and Pharmacotherapy, University of Szeged, Hungary.
Kovács
Péter P
PP
Kristóf
Attila A
AA
Nagy
Norbert
N
Tombácz
Dóra
D
Tóth
Judit S
JS
Ordög
Balázs
B
Jost
Norbert
N
Virág
László
L
Papp
Julius G
JG
Varró
András
A
Tóth
András
A
Boldogkoi
Zsolt
Z
D000818:Animals; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D048430:Cell Shape; D002470:Cell Survival; D002478:Cells, Cultured; D004285:Dogs; D031541:Fluorescence Resonance Energy Transfer; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D011558:Herpesvirus 1, Suid / Q000235:genetics*; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D008856:Microscopy, Fluorescence; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism / Q000502:physiology* / Q000821:virology; D018408:Patch-Clamp Techniques; D019209:Troponin C / Q000096:biosynthesis / Q000235:genetics*
D008164:Luminescent Proteins; D019209:Troponin C; D002118:Calcium