Herpesvirus-Mediated Delivery of a Genetically Encoded FluorescentCa2+Sensor to Canine Cardiomyocytes

Abstract

We report the development and application of a pseudorabies virus-based system for delivery of troponeon, a fluorescentCa2+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 (Ito), kinetics of the intracellularCa2+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 theItocurrent was significantly changed and no major shifts occurred in parameters of[Ca2+]itransients. Furthermore, we demonstrated that infection of cardiomyocytes with the virus did not affect the morphology, viability, and physiological attributes of cells.